Drug Development and Industrial Pharmacy

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Formulation development and in vitro evaluation

of gentamicin sulfate-loaded PLGA nanoparticles
based film for the treatment of surgical site
infection by Box–Behnken design

Chetan Dhal & Renuka Mishra

To cite this article: Chetan Dhal & Renuka Mishra (2019) Formulation development and in�vitro
evaluation of gentamicin sulfate-loaded PLGA nanoparticles based film for the treatment of surgical
site infection by Box–Behnken design, Drug Development and Industrial Pharmacy, 45:5, 805-818,
DOI: 10.1080/03639045.2019.1576719

To link to this article: https://doi.org/10.1080/03639045.2019.1576719

Accepted author version posted online: 30

Jan 2019.
Published online: 14 Feb 2019.

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DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY
2019, VOL. 45, NO. 5, 805–818
https://doi.org/10.1080/03639045.2019.1576719

RESEARCH ARTICLE

Formulation development and in vitro evaluation of gentamicin sulfate-loaded

PLGA nanoparticles based film for the treatment of surgical site infection by
Box–Behnken design
Chetan Dhal and Renuka Mishra
Department of Pharmaceutics and Pharmaceutical Technology, Institute of Pharmacy, Nirma University, Ahmedabad, Gujarat, India

ABSTRACT ARTICLE HISTORY

Objective: Gentamicin sulfate (GS)–loaded poly lactic-co-glycolic acid (PLGA) polymeric nanoparticles Received 14 May 2018
(PNPs) were developed and incorporated in film for the treatment of surgical site infection (SSI). Revised 28 December 2018
Method: PNPs were prepared by double emulsification solvent removal technique using ethyl acetate Accepted 24 January 2019
solution containing PLGA and polyvinyl alcohol (PVA) as an emulsifier. The emulsion was re-emulsified
KEYWORDS
using Gum Kondagogu (GKK). PNPs loaded film was prepared with 5% w/v solution of pullulan in PNPs Surgical site infection;
using solvent casting technique. Design of Experiment (DoE) study using Box–Behnken design was per- gentamicin sulphate;
formed for the optimization of PNPs. Drug release study was carried out for PNPs at phosphate buffer controlled release
saline (PBS) pH 6.4 and simulated wound fluid (SWF) pH 7.4. nanoparticles; PLGA; gum
Result: PNPs were found to have average particle size 280 ± 12.04 nm, polydispersity index (PDI) kondagogu; pullulan; film
0.15 ± 0.01 and zeta potential – 4.9 ± 0.84 mV. Scanning electron microscopy (SEM) showed spherical
nature of PNPs along with particle size of 160 ± 35.30 nm confirmed with transmission electron microscopy
(TEM). PNPs were found to be effective against Pseudomonas aeruginosa (PA) and Staphylococcus aureus
(SA). Optimized batch of film showed in vitro disintegration time below 8 min with tensile strength (TS)
0.06 ± 0.03 N/cm2 and percentage elongation (% E) 70.95 ± 4.29. X-ray diffraction study (XRD) confirmed
amorphous nature of GS, PLGA, pullulan, GKK and film.
Conclusion: PNPs showed controlled release of GS after an initial burst release. Developed film can be an
effective approach for management of SSI and control of antibiotic induced drug resistance.

Introduction essential medicine for the treatment of Gram-positive and Gram-

Wound healing involves interactions at cellular and molecular
level in a well-defined manner. The steps include inflammation,
cell migration, cell proliferation and tissue remodeling [1]. The
duration of these phases may vary from few weeks to months
depending upon the type of wound [2,3]. Surgical site infection
(SSI) is a pathological condition observed after surgery. In the
patients with SSI, prolonged inflammatory phase is observed due
to cellular shock subsequent to incision. SSI is associated with
microbial growth and biofilm formation, with more than 1000 col-
ony-forming units at the site of infection [4,5]. Global researches
have shown that some of the microbial strain coexisting with
highest probability are Staphylococcus aureus (SA), Pseudomonas
aeruginosa (PA), Escherichia coli and other associated strains
include Corneybacterium, Peptoniphilis, Serratia marcences,
Anaerococcus sp, Finegolida magna, Coagulase negative strepto-
cocci, Methicillin resistant S. aureus (MRSA) [6–11].
As per Centers for Disease Control and Prevention guidelines,
the management of SSI involves pre-operative, intra-operative,
and postoperative management. Gentamicin sulfate (GS) is
injected prophylactically to maintain therapeutic levels of drug
during surgical procedures [12]. Oral consumption of aminoglyco-
sides, such as GS and vancomycin, is preferred alternative for pre-
venting SSI after surgery. GS has been approved by WHO as an
negative infections [13].
Conventional dosage forms have limitations due to high fre-
quency of administration and development of antimicrobial resist-
ance. The alternative to the same is local therapy in the form of
antiseptic impregnated drapes, antimicrobial coated gauge, and
antibiotic creams. Several researchers have used collagen-based
GS implant/sponge (GCIS) after surgical procedures. Almedia et al.
reported no SSI in patients receiving GCIS compared to control
group [14]. Brehant et al. through research finding proved that
application of GCIS reduced SSI in patients upto 9% after colorec-
tal surgery [15]. Benedetto et al. found that out of 329 patients
receiving GCIS only 0.6% patients showed the deep sternal wound
infection (DSWI) and out of the rest; 2.01% patients showed symp-
toms of DSWI [16]. It was observed and reported that delivering
GS prophylactically after surgical procedure at the surgical site sig-
nificantly reduces the number of patients reported with
SSI [17–20].
Poly lactic-co-glycolic acid (PLGA) has been approved by
United States Food and Drug Administration as biodegradable
and biocompatible polymer with wide range of erosion times.
These properties makes it suitable controlled release polymer
[21–23]. Several researchers have successfully prepared PLGA poly-
meric nanoparticles (PNPs) of water soluble and insoluble drugs
by different techniques. Smaller size PNPs can be prepared with

CONTACT Renuka Mishra renukasharma81@rediffmail.com Department of Pharmaceutics and Pharmaceutical Technology, Institute of Pharmacy, Nirma
University, Ahmedabad, Gujarat, India
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
806 C. DHAL AND R. MISHRA
other polymers such as chitosan and alginate, but they lack con-
trolled release property. Chitosan and/or alginate PNPs require
chemical modification to provide controlled release of drug. The
maximum duration of drug release achieved with chemically
modified PNPs containing chitosan and alginate is less (upto
6 days) compared to PLGA [24–28]. Thus, PLGA was selected as
polymer for the preparation of PNPs. Gum Kondagogu (GKK), an
exudate from the bark of Cochlospermum gossipium is a nontoxic
polysaccharide. Chemically, GKK is composed of arabinose, galact-
ose, glucose, mannose, rhamnose, glucuronic acid, and galactour-
onic acid [29–31]. The emulsification property of GKK for the
preparation of liquid paraffin emulsion was evaluated between
concentrations 0.1% and 0.6% [32].
The objective of present investigation was development of GS-
loaded PLGA-based PNPs coated with GKK for the treatment of
SSI. Screening study using Plackett–Burman design and Design of
Experiment (DoE) study using Box–Behnken design were used for
the fabrication of PNPs. The PNPs were loaded in pullulan-based
film. Semisolid dosage forms, such as creams and gels, have limi-
tation of uneasiness and discomfort at the site, messiness, and
spread. Thus, film was selected due to convenience and accept-
ability as compared to conventional forms for application at the
surgical site during closure of the surgical incision. The PNPs and
film were evaluated for various parameters such as estimation of
particle size and polydispersity index (PDI), zeta potential, surface
morphological estimation, in vitro drug dissolution study, anti-
microbial effectiveness study, residual solvent analysis, in vitro dis-
integration study of film, drug content in film, estimation of
mechanical properties of film and X-ray diffraction study (XRD).
Material and methods
Materials
Gift sample of GS, PLGA 50:50 (Resomer RG 502) having molecular
weight 7 to 17 KDa and GKK were received from Jawa
Pharmaceuticals (Gurgaon, India), Evonik Industries (Evonik
Industries, Essen, Germany) and Nutrioma Healthcare (Hyderabad,
India), respectively. Sodium chloride, potassium chloride, sodium
hydrogen carbonate, di-sodium hydrogen orthophosphate, potas-
sium dihydrogen orthophosphate, sodium di-hydrogen orthophos-
phate, polyvinyl alcohol (PVA) having molecular weight 85 to
124 kDa were purchased from CDH Laboratories Pvt. Ltd. (New
Delhi, India). Nutrient agar media, nutrient broth and cellulose-
based dialysis membrane LA401 with molecular weight cutoff
12–14 KDa were procured from Himedia Laboratories Pvt. Ltd.
(Mumbai, India). SA (Microbial Type Culture Collection (MTCC
6538) and PA (MTCC 9047) were procured from Council for
Scientific and Industrial Research (CSIR) - Institute of Microbial
Technology (Chandigarh, India).
Methods
Physical compatibility study
During pharmaceutical processing or storage, physical interaction
may occur which can affect stability of the formulation. Physical
compatibility between GS and PLGA was performed by keeping
10 mg each of GS, PLGA and their physical mixtures in the ratio
1:1 in Biological Oxygen Demand (BOD) incubator (MOSIV – 3,
Sonar, New Delhi, India) at 40
C for 21 days [33–34]. The sample
were then analyzed using differential scanning calorimetry (DSC)
(DSC-6000, Perkin Elmer, MA).
Nanoparticle fabrication
PNPs were prepared by water-in-oil-in-water (w/o/w) double emul-
sion solvent removal method. Briefly, 50 mg/ml aqueous solution
of GS was prepared in double-distilled water (DDW). PLGA, in dif-
ferent GS to PLGA ratio was dissolved in fixed volume of ethyl
acetate with the help of a mechanical stirrer (2 MLH, Remi labora-
tory Instruments, Mumbai, India). Primary emulsion was prepared
by ultrasonicating the organic phase with an aqueous phase in
the presence of PVA as an emulsifier using Vibra Cell probe soni-
cator (Sonics and Materials Inc., CT) for 2 min. The primary emul-
sion was re-emulsified in GKK solution followed by particle
hardening and ethyl acetate removal. The PNPs were collected by
centrifugation at 10,000 rotation centrifugal force (rcf) for 20 min
using cooling centrifuge (Remi Pvt. Ltd., India). PNPs were washed
with DDW and lyophilized using lyophilizer (Delvac, Tamilnadu,
India) for storage and further studies. Lyophilization can increase
long-term storage of polymeric nanoparticles [34]. The lyophilized
PNPs were stored in microcentrifuge tubes sealed with parafilm
at 20
C.
Factors involved in the fabrication of PNPs were screened by
Plackett–Burman design using Minitab (Version 17, Minitab Inc.,
USA). Based on the results of the Plackett–Burman design, three
significant factors were identified. Each factor was considered at
two levels before applying DoE study using Box–Behnken design.
Preparation of films
Film was prepared by solvent casting method. Trial batches
included formulation of pullulan-based film at 2% and 5% w/v
concentration. To an aqueous solution of pullulan, sufficient
amount of plasticizer polyethylene glycol 400 (PEG 400) or propyl-
ene glycol (PG) were added individally. Before pouring the solu-
tion into teflon petri-plate, the solution was stirred using
magnetic stirrer (Remi, Delhi, India) for 1 h at 100 rpm. Films were
dried at 40
C in hot air oven (EIE instruments Pvt. Ltd.,
Ahmedabad, India) for 36 h. PNPs containing films were prepared
by dissolving required amount of pullulan and plasticizer in the
aqueous solution of PNPs. The resulting dispersion was stirred for
1 h at 100 rpm. The solution casting and drying conditions were
same as described previously.
Evaluation of PNPs and film
Percentage yield (PY). The PY was calculated as the percentage of
the weight of prepared PNPs to the total amount of components
(GS, PLGA, PVA and GKK) used in the process as shown in
Equation (1).
Weight of PNPs prepared
PY ¼  100 (1)
Total weight of components
Mean particle size and PDI. The mean particle size (d.nm) and PDI
were measured using four-side open disposable cuvette and zeta
potential (mV) was measured using zeta potential measuring cell
using particle size analyzer (Horiba India Pvt. Ltd, Bangalore) in
duplicate involving dynamic light scattering (DLS) technique.
Evaluation of surface morphology
The morphological evaluation of the lyophilized PNPs was carried
out by scanning electron microscopy (SEM) (Carl Zeiss Evo 18 SEM
with EDXRF, Jena, Germany) and transmission electron microscopy
(TEM) (FEI Tecnai 20 Hillsboro, USA). For SEM, the PNPs were

coated with gold layer which imparts sufficient conductivity to the
particles before being examined. Double side adhesive carbon
tape was placed on the sample mount. Gold-layered PNPs were
placed on the upper side of carbon tape and sample mount was
placed in the holder to capture SEM micrographs. For TEM, PNPs
were dispersed in DDW and placed on the coated copper grids
with mesh size 60 and allowed to dry in air. The copper grid was
placed on the grid holes, locked with locking flange in the piezo
holder and TEM images were recorded.
Drug loading (DL) and entrapment efficiency (EE) in PNPs. DL is
defined as the amount of drug to amount of lyophilized PNPs pre-
pared and EE is defined as the amount of drug entrapped in
nanoparticles over total amount of drug taken.
For the estimation of DL, 50 mg lyophilized PNPs were sus-
pended in a mixture containing 0.5 ml DDW and 0.5 ml ethyl acet-
ate and vortexed for 1 min (Cyclomixer CM-101, Remi, Delhi,
India). The aqueous layer of the solution was used to estimate the
content of GS in duplicate.
After centrifugation process, the pellet and centrifuged solu-
tion were separated. This centrifuged solution here is referred to
as supernatant. The pellet was washed with DDW and centri-
fuged again. The resulting liquid after re-centrifugation is
referred as wash medium. The two solutions were combined and
used for the estimation of EE. The estimation was performed
in duplicate.
The amount of GS was estimated by validated derivatization
method suggested by Kowalczuk et al. with minor modifications
[35]. Calibration curve for GS was plotted from 1.6 mg/ml to
25.6 mg/ml concentration and the value of correlation coefficient
(R2) was found to be 0.99. Derivatization reagent was prepared by
dissolving 20 mg of o-phthalaldehyde (OPA) in 1.0 ml methanol
followed by addition of 0.1 ml thioglycolic acid and diluting the
solution to 10 ml with 0.20 M boric acid solution adjusted to pH
10 with 40% sodium hydroxide solution. 2.50 mM hydroxy propyl
– b – cyclodextrin solution was used as reaction stabilizer. The GS-
OPA complex was prepared by reacting a mixture containing
0.50 ml sample solution, 0.50 ml derivatization reagent and 1.50 ml
stabilizer at 60
C for 15 min followed by cooling to room tem-
perature and estimated using UV-Visible spectrophotometer
(Jasco, Mumbai, India) at 335 nm. Percentage EE (% EE) and per-
centage DL (% DL) were calculated by Equations (2 and 3) [36].
Total drug ðmgÞFree drug ðmgÞ
% EE ¼  100 (2)
Total drug ðmgÞ
Total drug ðmgÞFree drug ðmgÞ
% DL ¼  100 (3)
Total weight of PNPs ðmgÞ
In vitro dissolution study. In vitro dissolution study was performed
using dialysis bag (LA 401, Himedia, Mumbai, India) in two differ-
ent dissolution media, namely phosphate buffer saline (PBS) pH
6.4 and simulated wound fluid (SWF) pH 7.4. During surgical pro-
cedure, incision disrupts skin pH and shifts it to pH above 6 pro-
viding rationale for the selection of PBS pH 6.4 as dissolution
medium [37]. PBS pH 6.4 was prepared as per method specified in
Indian Pharmacopoeia 2010 by dissolving 1.79 g of disodium
hydrogen orthophosphate, 1.36 g of potassium dihydrogen ortho-
phosphate and 7.02 g of sodium chloride in 1000 ml of DDW [38].
Chronic wounds exist at pH range of 7.2 to 8.9 providing rationale
for selection of SWF pH 7.4 [39]. SWF pH 7.4 was prepared by dis-
solving 0.68 g sodium chloride, 0.22 g potassium chloride, 2.5 g
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 807
sodium hydrogen carbonate and 0.35 g sodium di-hydrogen phos-
phate in 100 ml DDW, adjusted to pH 7.4 [40]. Prior to the study,
the dialysis bag was treated as per the procedure specified by
Himedia [41] and stored in dissolution media.
After centrifugation, the supernatant was separated and PNPs
in the form of viscous liquid were collected in a microcentrifuge
tube. From the microcentrifuge tube, 2 ml PNPs containing viscous
liquid was transferred into the dialysis bag, tied and suspended in
200 ml dissolution medium maintained at 37
C. Dissolution
medium equivalent to 2 ml was withdrawn at predetermined time
points (0 h, 0.016 h, 0.083 h, 0.16 h, 0.5 h, 1 h, 6 h, 10 h, 24 h, 48 h,
72 h, 96 h, 120 h, 144 h, 168 h, 192 h and 216 h) and replenished
with fresh medium in order to maintain sink condition. Estimation
of GS was performed in duplicate by the method described earlier
for the estimation of DL at 335 nm.
Antimicrobial effectiveness study. Identification of the zone of
inhibition (ZOI) with PNPs was carried out as per procedures
specified in Indian Pharmacopoeia for antimicrobial assay with
slight modification [38]. GS equivalent to 25 mg/ml concentration
was prepared from stock solution of PNPs and marked as S3 (S3
represents the median level concentration). Other dilutions con-
taining GS equivalent to 16, 25, 31.20 and 39.05 mg/ml were pre-
pared from stock solution and marked as S1, S2, S4 and S5,
respectively. The concentrations were selected such that the ratio
between each lower and higher level remained 4:5. Nutrient agar
plates containing microbial culture (1 ml) were prepared and 4
wells were made in each petri-plate using 0.6-mm stainless steel
borer. Wells were filled with 0.5 ml PNPs solution in a manner that
each alternate well in every petri-plate received S3 and other wells
received S1, S2, S4, and S5, respectively, in duplicates. The ZOI
was measured in the petri-plates after an incubation for 24 h at
37
C in BOD incubator (Samiksha Industrial Corporation, Mumbai,
India). A graph between square of ZOI versus log concentration
was plotted and MIC was estimated by taking antilog of the con-
centration obtained by extrapolating the curve till it intersected
the x-axis.
For the measurement of MIC using nutrient broth, a stock solu-
tion of GS and GS equivalent in PNPs was prepared by dissolving
sufficient weight in DDW to obtain a concentration of 1000 mg/ml
for GS and 300 mg/ml for GS equivalent in PNPs. From the stock
solution, sufficient volume was pipetted in different test tubes
containing 0.5 ml microbial culture and diluted to 10 ml with nutri-
ent broth to obtain concentration between 0.06 to 123 mg/ml and
1.92 to 61.44 mg/ml for GS and GS equivalent in PNPs, respectively.
The test tubes were incubated for 24 h at 37
C in BOD incubator
and evaluated for the presence of growth.
Estimation of residual solvents in PNPs. Ethyl acetate was used as
one of the components during fabrication of PNP. It is a class III
solvent having acceptable limit below 5000 mg/ml as per ICH Q3C
(R5) guideline [42]. The level of ethyl acetate was estimated by
gas chromatographic analysis performed on Agilent Gas chro-
matograph equipped with head space (GC-HS) (Agilent
Technologies 7890 A, New Delhi, India) using Agilent capillary col-
umn, DB-624, with dimensions 30 m  0.25 mm  1.4 mm, Nitrogen
as carrier gas maintained at a flow rate of 0.50 ml/min with 500 ml
as injection volume. The run time was kept constant for 45 min.
Sufficient amount of lyophilized powder of PNPs was dispersed
in carrier solvent, NN0 -dimethyl formamide and injected in tripli-
cate. The chromatogram was compared with standard chromato-
gram of ethyl acetate. Concentration of ethyl acetate in PNPs was
calculated using Equation (4).
808 C. DHAL AND R. MISHRA
Table 1. Levels of independent variables selected for plackett burman design.
Parameter Code Independent variables selected
A Type of PLGA (Categorical)
B Volume of Ethyl acetate (ml)
C Quantity of PLGA(mg)
D Concentration of Primary emulsifier (%)
E Duration of Sonication (min)
F Temperature while sonication (Categorical)
G Concentration of Secondary emulsifier (%)
H Volume of secondary emulsifier (ml)
I Speed of stirring (rpm)
J Duration of stirring (h)
Concentration of organic solvent ðlg=mlÞ
Area of standard solvent Dilution of test
¼  (4)
Area of test solvent Dilution of standard
 Potency of standard  1000
In vitro disintegration study. It was performed by placing
1  1 cm2 film in a petri-plate containing 10 ml SWF. The time
taken for the disintegration of film was measured in duplicate and
considered as in vitro disintegration time.
Drug content in film. A portion of film having dimension 2  1 cm2
was cut, weighed, and dissolved in 1 ml DDW and vortexed with
an equal volume of ethyl acetate. From the vortexed solution,
0.50 ml of aqueous medium was used for the estimation of GS in
film. The drug content was measured in duplicate as per the
method described in the section of measurement of % DL.
Mechanical properties of film. Mechanical properties of film
namely tensile strength (TS) and % elongation (% E) were esti-
mated in duplicate using Digital Tensiometer (EIE Instruments,
Ahmedabad, India). Film with dimensions 1.5  7 cm2 free from
physical imperfection were cut and estimated for thickness using
Digital Thickness gauge (Mitutoyo Corporation, Kanagawa, Japan).
The film was held between two clamps of digital tensiometer.
One of the clamps was stationary and other clamp moved down
to pull the film at rate of 35 inch/min. The clamps were positioned
at a distance of 3 cm. Equations (5 and 6) were used to calculate
TS and % E, respectively.

Force required to break film ðkgÞ
TS N=cm2 ¼  9:8
Initial cross sectional area of the film ðcm2 Þ
(5)
Increase in length ðmmÞ
% E¼  100 (6)
Initial length of the film ðmmÞ
XRD of film and PNPs. XRD of GS, PLGA, GKK, optimized batch of
PNPs and optimized batch of film FF16 were performed using
Xpert MPD X-ray diffractometer (Philips, Holland) at 45 kV and
30 mA. It uses copper Ka X-ray radiation source and graphite
monochromator to produce X-rays of 1.5405 Ð. Sample was
placed in copper holder, placed above the sample tray and ana-
lyzed between 5
to 50
.
Statistical analysis
Statistical software, Minitab was used for screening of factors
using Plackett–Burman design and DoE study using Box–Behnken
design. The results were considered statistically significant when
the obtained p values was <0.05.
Level () Level (þ)
End capped Uncapped
5 15
75 (1:1.5) 225 (1:4.5)
0.5 3.5
2 5
Ice Bath RT
0.05 0.35
20 40
200 800
2 8
Results and discussion
Nanoparticle fabrication and evaluation
Major attention has been paid to PLGA PNPs over other polymer-
based PNPs due to its superior properties such as biodegradable
nature, controlled release, and noninteractive nature [21–23,43].
Water-soluble drugs tends to partition between solvents therefore
double emulsion solvent removal technique was adopted for the
fabrication of PNPs. Numerous process-related factors having
effect on DL including drug-to-polymer ratio, volume of solvent,
concentration of primary and secondary emulsifier, pH and ionic
strength of outer medium were considered. Several trial batches
were prepared prior to Plackett–Burman design. PLGA was solubi-
lized in two different organic solvents (ethyl acetate and dichloro-
methane), PVA was used at variable concentration between 2%
and 3.5% as primary emulsifier and emulsification was done by
ultrasonication using probe sonicator at different amplitude 20%,
30%, and 40%. For the preparation of secondary emulsion, effect
of GKK concentration (0.05% to 0.35%), pH (acidic, basic, and
unaltered pH of GKK solution), and addition of salt
were considered.
Ethyl acetate is partially miscible with water and molecular dis-
persion between ethyl acetate and PLGA yields higher DL [44,45].
PNPs prepared with dichloromethane had higher DL and smaller
particle size in comparison to PNPs prepared using ethyl acetate.
PLGA possess glass transition close to 50
C. Heat generation was
observed during ultrasonication process. Lower DL was observed
with batches prepared at high amplitudes. Lower DL was seen in
the PNPs prepared after adding salt to the solution of GKK. Acidic
pH showed lower DL, however lower PY was observed with basic
pH. Unaltered pH of GKK solution was further selected. Based on
these observations, Plackett Burman design was applied for
screening of 10 significant factors (2 categorical and 8 numerical)
each considered at 2 levels as summarized in Table 1.
From the results of Plackett–Burman design as summarized in
Table 2, it can be seen that size of PNPs varied in the range
30 nm to 438 nm with PDI ranging between 0.51 and 9.41.
Although the usual PDI range is between 0 to 1; however, PDI
more than 1 was observed with batches FP1, FP3-FP5, FP7-FP8,
and FP10-FP12 indicated particles of improper shape. Thus, condi-
tion for preparation of these batches were not acceptable.
Batches FP1 and FP3 were found to have lower DL (<20%), higher
PDI (>9.0) and lower particle size (30 nm). Batches FP4, FP8,
FP10, and FP12 depicted % DL between 20% and 80% with par-
ticle size more than 250 nm and PDI in the range 1.0–2.0. Batch
FP5, FP7, and FP11 showed an inverse relation between particle
size and PDI. It was also seen with these batches that smaller par-
ticles possessed higher % DL. Acceptable PDI and particle size was
observed with batches FP2, FP6, and FP9. Average particle size
was close to 250 nm with PDI between 0.50 and 0.70. Level of sig-
nificance of individual factor was determined on the basis of ‘p’
 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 809

Figure 1. Pareto charts and Main effects plots in Plackett Burman design generated for (A) % EE, (B) % DL, (C) Particle size and (D) PDI.
810 C. DHAL AND R. MISHRA

Figure 2. Contour plot for (A) % DL, particle size and PDI and interaction plots for (B) % DL, (C) particle size and (D) PDI.

Table 2. Composition of batches FP1 to FP12 using Plackett–Burman design and evaluation results.
Batch no. A B (ml) C (mg) D (%) E (min) F G (%) H (ml) I (rpm) J (h) % EE % DL P. size (nm) PDI
FP 1 E 15 75 3.5 5 I 0.35 20 200 2 45.03 2.05 30 9.06
FP 2 U 15 225 0.5 5 I 0.05 20 800 8 78.96 2.62 297.7 0.51
FP 3 U 15 75 0.5 2 R 0.35 40 200 8 0.00 1.73 19 9.41
FP 4 U 5 225 3.5 5 I 0.35 40 200 8 27.60 30.67 358 1.72
FP 5 E 15 225 0.5 5 R 0.05 40 200 2 35.46 8.18 136.4 2.48
FP 6 E 5 225 3.5 2 R 0.05 20 200 8 53.30 33.54 264.2 0.69
FP 7 U 15 225 3.5 2 R 0.35 20 800 2 39.78 33.25 26.8 9.20
FP 8 E 5 225 0.5 2 I 0.35 40 800 2 12.38 77.16 438 1.59
FP 9 U 5 75 0.5 2 I 0.05 20 200 2 64.55 2.50 289 0.57
FP 10 E 5 75 0.5 5 R 0.35 20 800 8 11.31 54.04 329 1.28
FP 11 U 5 75 3.5 5 R 0.05 40 800 2 54.64 1.33 270.4 1.84
FP 12 E 15 75 3.5 2 I 0.05 40 800 8 90.10 22.40 266.6 1.47
Type of PLGA; E ¼ End capped and U ¼ Uncapped.
Temperature condition during primary emulsificationl; I: Ice bath and R: Room temperature.
P: size refers to particle size.

value. Pareto charts and main effects plot were obtained using
Minitab software for % EE, % DL, particle size, and PDI, respect-
ively, as shown in Figure 1 (A–D). Comparing the effects of all fac-
tors, factors B, D, and G were found significant (p < 0.05).
Main effects plots suggest the effect of different factors on
PNPs. Type of PLGA showed an effect on PDI, whereas % DL, %
EE, and particle size remained unchanged. Volume of ethyl acetate
used to solubilize PLGA had a drastic effect on % DL, particle size,
and PDI. Lower volume of ethyl acetate was found beneficial for
PNPs fabrication. Higher amount of PLGA for PNPs preparation
yielded higher % DL, lower PDI, whereas % EE and particle size
were less affected. With higher concentration of PVA, % DL, % EE,
and PDI increased and particle size decreased. Duration of sonic-
ation did not show much effect on the PNPs. Ice bath yielded
PNPs with higher % DL, % EE and particle size and lower PDI.
However, higher concentration of GKK favored DL and particle
size with lower % EE and higher PDI. Volume of GKK solution did
not show much change on the response parameters. Speed and
duration of stirring showed similar responses, that is, increasing
the speed and duration yields PNPs with higher % EE, % DL, par-
ticle size, and lower PDI.
Further, DoE study using Box–Behnken design was performed
for three factors, namely volume of ethyl acetate, concentration of
primary emulsifier, and concentration of secondary emulsifier each
considered at two levels. As summarized in Table 3, 15 batches
FB1 to FB15 were prepared.
All the prepared batches had particle size ranging between
214 nm to 348 nm and PDI above 0.20. Batches FB1, FB3, FB6, FB7,
FB8, FB13, and FB15 showed % DL above 30%, whereas batches
FB5, FB9, FB10, FB11, and FB12 showed % DL between 10% and
 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 811

30%. Less than 10% DL was found for batches FB2, FB4, and FB14. GG significantly affect (p < 0.05)% DL, particle size, and PDI.
Based upon the observations, using Minitab software design space
X1 ¼  24:2 þ 48:01 B þ 56:1 D þ 370 G
(Figure 2(A)) and interaction plots were generated in support of
optimization (Figure 2(B–D)).  1:735 BB  15:29 DD  831 GG (7a)
The interaction plot specifically suggests the effects of all fac-  0:50 BD  26:5 BG þ 91:6 DG
tors on % DL, particle size, and PDI. However, lower concentration
X2 ¼ 0:160 þ 0:0401 B þ 0:013 D  0:14 G
of primary emulsifier (0.5%) was noninteracting near the desired
ranges of particle size (X1), PDI (X2), and % DL (X3). Polynomial þ 0:00043 B  B þ 0:0490 D  D þ 4:67 G  G (7b)
mathematical expression was also generated for X1, X2 and X3 as  0:0220 B  D  0:023 B  G  0:548 D  G
given in Equation (7(a)–(c)). From the equations, it was found that X3 ¼ 21:9 þ 6:1 B  37:3 D  261 G
factors B, D, G, and their polynomial interactions BB, DD, and
 0:222 BB þ 8:77 DD þ 926 GG  0:72 BD (7c)
Table 3. Composition of batches FB1 to FB15 for DoE study using box behnken  2:8 BG þ 76:8 DG
design and evaluation results.
Response surface curve for % DL, particle size, and PDI were
Batch no. B (ml) D (%) G (%) % DL Particle Size (nm) PDI
generated using Minitab software (Figure 3 (A–C)). It depicts the
FB 1 5 3.5 0.2 31.7 250.8 0.41 correlation between one of the response factors and two varia-
FB 2 5 2 0.05 1.01 226.2 0.46
FB 3 15 2 0.35 58.68 306 0.30 bles. Better % DL was observed with higher level of factor D and
FB 4 10 0.5 0.05 6.36 326 0.52 lower level of factor G. Factor B did not show an effect on % DL.
FB 5 10 2 0.2 16.63 348 0.28 Desirable particle size was obtained with higher level of D and
FB 6 5 0.5 0.2 37.47 214 0.25
FB 7 5 2 0.35 62.18 280.6 0.47
lower levels of B and G. Desired lower PDI value was achieved
FB 8 10 3.5 0.35 42.56 305 0.23 with an increase in factors B, G and D close to 0.2%. It was
FB 9 10 3.5 0.05 24.07 305 0.21 observed that during particle hardening, ethyl acetate displaced
FB 10 10 2 0.2 16.63 348 0.28 from PNPs. Therefore, volume of ethyl acetate is critical during
FB 11 15 3.5 0.2 13.37 319 0.22
FB 12 10 2 0.2 16.63 348 0.28 fabrication process. Optimized batch of PNPs (batch F2) was
FB 13 15 0.5 0.2 40.72 297.1 0.72 selected on the basis of composite desirability (D) value, 0.5798
FB 14 15 2 0.05 5.83 331 0.36 from response optimizer plot (Figure 4). The composition for
FB 15 10 0.5 0.35 55.77 243.6 1.03
batch F2 is summarized in Table 4.

Figure 3. Response surface plots generated using Box–Behnken design for (A) % DL, (B) particle size and (C) PDI.
812 C. DHAL AND R. MISHRA
Figure 4. Composite desirability and optimization condition obtained using
Box–Behnken design.
Physical compatibility study
Figure 5(A) shows the DSC thermograms for PLGA, GS, and phys-
ical mixture. The thermogram of PLGA (Thermogram A) illustrates
glass transition temperature (Tg) at 37
C confirming its amorph-
ous and glassy nature with rigid chain structure [22,46]. Melting
was observed at peak temperature 50.46
C for PLGA.
Thermogram B exemplifies DSC of GS and two endothermic peaks
were observed. Similar to the description given by Rosenkrantz
et al. broad endothermic peak from 47.11
C to 140.01
C was
observed with its peak at 90.60
C due to loss of water. A sharp
endothermic peak at 244.13
C corresponding to melting decom-
position of gentamicin was also observed [47]. Thermogram C
embodies 1:1 physical mixture of GS and PLGA. Intact peaks for
PLGA and GS were observed at 47.3
C and 247.37
C, respectively,
which confirm the physical compatibility between them.
Percentage yield and particle size estimation
The PY for the F2 batch of PNPs was 77.68 ± 5.60%. The mean par-
ticle size estimated by DLS technique was found to be
280 ± 12.04 nm. Based on the PDI value 0.15 ± 0.01, it was con-
firmed that the PNPs were monodispersed. A 0.20% w/v aqueous
solution of GKK used as secondary emulsifier possessed
42.4 ± 1.64 mV charge. Naidu et al. demonstrated that GKK pos-
sess high negative zeta potential at low concentration which may
be attributed due to the presence of large number of carboxylic
acid and hydroxyl groups [48]. The zeta potential possessed by
PNPs was found to be 4.9 ± 0.84 mV. The difference in the result-
ant potential after electrostatic interaction is approximately
>þ35 mV, it confirms the stability of the solute particles of pri-
mary emulsion.
Table 4. Composition of batches F1 and F2 for in vitro dissolution study.
Batch
Components F1 F2
PLGA (Uncapped) (mg) 200 200
Ethyl acetate (ml) 7.37 8
GS solution (mg/ml) 50 50
Conc of PVA (%) 3.48 3
Duration of sonication (min) 2 2
Temperature while sonication (Ice bath) (oC) 8 8
Conc of secondary emulsifier (%) 0.2 0.2
Volume of secondary emulsifier (ml) 30 30
Speed of stirring (rpm) 800 800
Duration of stirring (h) 8 8
Morphological characterization of PNPs
SEM micrographs of gold-plated PNPs from batch F2 were cap-
tured at 6.33 KX magnification. PNPs were spherical, possessed
smooth regular surface but were fused with each other. This could
be attributed to the presence of cohesive forces between the
layers of GKK over the surface of PNPs (Figure 5(B)). TEM of the
batch F2 confirmed the spherical nature of particles with average
particle diameter 160 ± 35.30 nm (Figure 5(C). The particle size of
PNPs measured by DLS technique is higher than that measured
with TEM study. The difference can be explained by the fact that
TEM measures the actual diameter, whereas hydrodynamic diam-
eter is measured with DLS technique. Also, when PNPs are sus-
pended in a solvent, there is a tendency of adherence of thin
electric dipole layer of the solvent over the particles [49].
% DL and in vitro dissolution study
The in vitro dissolution study was performed for two batches, F1
and F2. F1 indicates batch selected from design space generated
with Minitab software, whereas F2 refers to the optimized batch.
The composition of batches F1 and F2 are summarized in Table 4.
Before transferring the PNPs in the form of viscous liquid into the
dialysis bag, amount of GS was estimated. %DL for the batches F1
and F2 was found to be 48 and 60%w/w, respectively.
Figure 5(D) shows the cumulative percentage drug release for
batches F1 and F2 up to 216 h in PBS pH 6.4 and SWF pH 7.4.
Sustained release of GS was observed from PNPs. The release
from PNPs occurs due to hydrolytic cleavage of the linkage
between lactic acid and glycolic acid in PLGA mediated through
steps such as diffusion, solvent penetration, degradation and ero-
sion [50]. PLGA is known to show initial burst release [22].
However, it can be controlled by re-emulsifying the PLGA matrix
with other polymers such as natural gums, poly-caprolactone
(PCL), and PVA [50]. Drug release for batch F1 at 1 h, 6 h and 24 h
was 13.61 ± 0.40%, 19.48 ± 0.27%, and 25.43 ± 0.33% using PBS pH
6.4 and 24.48 ± 0.01%, 29.27 ± 0.10%, and 38.9 ± 0.21% using SWF
pH 7.4, respectively. However, similar drug release of 83.7 ± 0.10%
and 84.4 ± 0.06% was observed at 216 h using PBS pH 6.4 and
SWF pH 7.4, respectively. Similar patterns of drug release for batch
F2 in PBS pH 6.4 and SWF pH 7.4 were also observed. Drug
release for batch F2 at 1 h, 6 h and 24 h was 11.85 ± 0.01%,
22.66 ± 0.36%, and 32.46 ± 0.22% using PBS pH 6.4 and
30.6 ± 0.4%, 34.3 ± 0.27%, and 48.4 ± 0.32% using SWF pH 7.4,
respectively. Drug release of 89.98 ± 0.15% and 90.1 ± 0.01% was
observed at 216 h using PBS pH 6.4 and SWF pH 7.4, respectively.
Both batches F1 and F2 showed initial drug release followed by
gradual controlled release up to 216 h indicating suitability for SSI
treatment. Higher drug release for batches F1 and F2 was
observed using SWF pH 7.4 compared to PBS pH 6.4. The reason

Figure 5. (A) Overlay DSC endotherm, surface morphological images of PNPs (B) SEM at 6.33 KX magnification, (C) TEM at 500 nm scale and (D) in vitro drug dissol-
ution study for batches F1 and F2.
for higher drug release at SWF pH 7.4 compared to PBS pH 6.4
could be due to property of PLGA. PLGA is more likely to show
less pronounced degradation at slight acidic pH and alkaline
media is favorable for degradation [22]. To confirm the effect of
pH of dissolution medium on dissolution profiles, t-test was
applied on the results of dissolution study for batches F1 and F2.
The values of t-stat and t-critical were generated by using
Microsoft excel 2013. The t-stat value and t-critical value of 6.72
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 813
and 2.11 for batch F1 and 6.96 and 2.11 for batch F2 respectively
was obtained. As the t-stat value > t-critical value, it was con-
cluded that pH of dissolution medium significantly affected drug
release. The dissolution study data was also statistically analyzed
using DD solver (Microsoft office 2013) for the determination of
dissolution models [51]. The drug release order was selected on
the basis of regression coefficient (r2) obtained by plotting graphs
for zero order, first order, Korsmeyer–Peppas, Higuchi matrix, and
814 C. DHAL AND R. MISHRA
Figure 6. Antimicrobial effectiveness study (A) graph between log concentration versus square of ZOI and (B) images of ZOI observed against SA and PA.
Hixon Crowell model. PNPs in PBS pH 6.4 followed Higuchi Matrix
model with r2 value 0.94 and 0.98 for batches F1 and F2, respectively,
whereas PNPs in SWF pH 7.4 followed Korsmeyer–Peppas model
with r2 value 0.97 and 0.96 for batches F1 and F2, respectively. In
Higuchi matrix model, the drug release depends on the concentra-
tion gradient of the solute molecules, whereas Korsmeyer–peppas
model clearly signifies release from polymeric systems [52]. Further,
the diffusion kinetics was estimated using Equation (8).
slope of the curve obtained with korsemeyer peppas model
n¼
2:303
(8)
where n represents a numerical factor correlated to type of diffu-
sion followed. If the value of n lies between 0 to 0.5, the release
is fickian, if the value of n lies between 0.5 to 1, the diffusion is
considered as nonfickian. The value for ‘n’ for the batches F1 and
F2, was found to be 0.23 and 0.27; 0.13, and 0.12 in dissolution
media PBS pH 6.4 and SWF pH 7.4, respectively indicating fick-
ian diffusion.
Antimicrobial effectiveness study
Figure 6(A) depicts the plot of log dose (log concentration) versus
square of ZOI and Figure 6(B) depicts the ZOI observed against SA
and PA, respectively. GS was found to be effective against SA and
PA. From the observations of antimicrobial study performed using
agar plate method, MIC for PNPs containing GS was found to be
6.84 mg/ml and 13.07 mg/ml for SA and PA, respectively. Table 5
summarizes the result of antimicrobial study against SA and PA
for different concentration of GS in free state and PNPs containing
GS. GS at concentration 15.36 mg/ml and 30.72 mg/ml showed
inhibitory activity against SA and PA, respectively. The effective
concentration of GS in free state and PNPs containing GS were
found to be similar for PA; however, PNPs containing GS showed
inhibition in growth of SA at much lower concentration compared
to GS in free state. The difference in effectiveness may be attrib-
uted to the surface charge of PNPs [53].
Estimation of residual solvents in the PNPs
Figure 7(A) shows standard solution of ethyl acetate has a reten-
tion time at 14.32 min. Figure 7(B) shows chromatogram for PNPs
a peak at retention time 14.34 min indicating the presence of
ethyl acetate in the PNPs. The amount of ethyl acetate was found
out to be 489 ± 0.28 mg/ml in the PNPs which is below the accept-
able limit, 5000 mg/ml as per ICH Q3C (R5) guidelines for residual
solvents [42]. The observed amount endorsed acceptable limit of
ethyl acetate as residual solvent in PNPs.
Preparation of film
Films using pullulan were prepared at varying concentration as
described in Table 6. Batches FF1, FF2 at 2% and 5% w/v pullulan,
 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 815

Table 5. Antimicrobial effectiveness study.

Incubation time (24 h)
Concentration (mg/ml) S. aureus P. aeruginosa
GS in free state
0.06 þ þ
0.12 þ þ
0.24 þ þ
0.48 þ þ
0.96 þ þ
1.92 þ þ
3.84 þ þ
7.86 þ þ
15.36  þ
30.72  
61.44  
122.88  
PNPs containing GS
1.92 þ þ
3.84 þ þ
7.86 – þ
15.36 – þ
30.72 – –
61.44  –
Media Control – –
Culture control þ þ

%E significantly increased. Compared to batch FF15, batch FF16

Figure 7. Chromatograms obtained from GC-HS analysis (A) Standard and
(B) PNPs.
showed higher %E but lower TS due to the presence of higher
amount of PG. Changes in the mechanical properties could be
due to plasticization effect by PG. Thus, batch FF16 was selected
as optimized batch due to highest % E and acceptable TS.

respectively, were stiff and brittle. These batches had an average
thickness 0.17 ± 0.01 mm and 0.26 ± 0.03 mm. Batch FF3 to FF10
containing plasticizer PEG 400 or PG were flexible, smooth with
better mechanical properties. The average thickness for the films
prepared with PG as plasticizer varied between 0.07 ± 0.03 mm to
0.17 ± 0.08 mm, whereas films prepared with PEG 400 as plasticizer
possessed thickness between 0.17 ± 0.02 to 0.24 ± 0.05 mm. Films
containing PG had lower thickness, thus PG was selected for the
preparation of PNPs loaded film FF13–FF16. The thickness of PNPs
incorporated batches FF13 and FF14 were found to be
0.15 ± 0.02 mm and 0.18 ± 0.06 mm, whereas the thickness of the
batches FF15 and FF16 was found to be between 0.27 ± 0.07 mm
and 0.24 ± 0.04 mm, respectively. Batches FF12, FF15, and FF16
had nearly 50% DL. Maximum % DL was observed with batch
FF16 and least loading was observed in batch FF13. Based on
these observations, batch FF16 was selected containing 5% w/v
pullulan and PG as plasticizer at 0.4:1 ratio (plasticizer: polymer).
Batches FF1 to FF10 prepared without PNPs showed rapid in
vitro disintegration time of less than 1 min in SWF. PNPs contain-
ing batches (FF11 to FF16) showed longer in vitro disintegration
time. Batches FF15 and FF16 showed maximum in vitro disintegra-
tion time of 6 min and 8 min, respectively this might be due to
the higher concentration of pullulan. Higher in vitro disintegration
time was desirable as it provided time for ease of application at
the surgical site. Thus, batches FF15 and FF16 were selected.
Estimation of mechanical properties of film
Batches FF11 to FF16 were evaluated for mechanical properties
using Digital Tensiometer. The observations are summarized in
Table 7. It was observed that, TS decreased and %E increased as
amount of PG increased. As concentration of pullulan increased,
X-ray diffraction study
Figure 8 shows X-ray diffraction pattern for GS, PLGA, pullulan,
GKK, optimized batch of PNPs, and optimized batch of film FF16.
Figure 8(A) depicts single sharp peak at 44.61
for GS. The pres-
ence of single sharp peak may be due to the recrystallization of
GS. Figure 8(B–D) shows no sharp diffraction peak, and thus, it
was established that PLGA, pullulan, and GKK exist in amorphous
form. Diffractogram of the optimized batch of PNPs indicated
sharp peaks at 9.57
, 20.25
, 25.01
, 35.95
, 40.18
, 43.35
and
44.95
(Figure 8(E)). The presence of peak at 44.95
indicated sur-
face associated GS in PNPs confirming loading of GS. No specific
peak was observed for optimized batch of film FF16 (Figure 8(F))
indicating its amorphous surface and incorporation of PNPs inside
the film and not over the surface of the film.
Conclusion
GS-loaded PLGA-GKK encapsulated PNPs were formulated for the
treatment of SSI at localized site. Controlled release of GS was
observed due to PLGA after an initial burst release. The burst
release is effective for the attainment of initial concentration of
GS at site of application. Batches F1 and F2 showed initial drug
release followed by controlled release up to 216 h indicating suit-
ability for SSI treatment. Drug release was affected by pH of the
dissolution medium, PBS pH 6.4 and SWF pH 7.4. The process
optimization included Plackett–Burman design for screening of
significant factors and DoE study using Box–Behnken design for
optimization. PNPs prepared with optimized batch had particle
size less than 280 nm and PDI close to 0.15 indicating monodis-
perse nature of the system. The PNPs possessed spherical and
smooth surface as confirmed by SEM and TEM study. Batch F2 of
PNPs showed fickian diffusion with drug release upto
816 C. DHAL AND R. MISHRA

Table 6. Composition of batches FF1 to FF16 and evaluation results.

Batch no. Pullulan (%) Plasticizer Plasticizer: Polymer Solvent (upto 30 ml) Drug Content (%) In vitro disintegration Time (min)
FF1 2 – – DDW – <1 min
FF2 5 – – DDW – <1 min
FF3 2 PEG 400 (0.2:1) DDW – <1 min
FF4 2 PEG 400 (0.4:1) DDW – <1 min
FF5 5 PEG 400 (0.2:1) DDW – <1 min
FF6 5 PEG 400 (0.4:1) DDW – <1 min
FF7 2 PG (0.2:1) DDW – <1 min
FF8 2 PG (0.4:1) DDW – <1 min
FF9 5 PG (0.2:1) DDW – <1 min
FF10 5 PG (0.4:1) DDW – <1 min
FF11 2 – – PNPs solution 31.45 ± 2.64% <2 min
FF12 5 – – PNPs solution 47.26 ± 3.51% <2 min
FF13 2 PG (0.2:1) PNPs solution 17.04 ± 1.25% <3 min
FF14 2 PG (0.4:1) PNPs solution 24.26 ± 3.55% <2 min
FF15 5 PG (0.2:1) PNPs solution 52.11 ± 1.01% <6 min
FF16 5 PG (0.4:1) PNPs solution 54.64 ± 0.98% <8 min

Figure 8. XRD diffractogram for (A) GS, (B) PLGA, (C) pullulan, (D) GKK, (E) Optimized batch of PNPs and (F) optimized film.

Table 7. Results of mechanical properties of batches FF11 to FF16. SRF fellowship to Mr. Chetan Dhal for performing the study as a
Batch No TS ± SD (N/cm ) 2
% E ± SD part of PhD research work to be submitted to Nirma University.
FF11 0.10 ± 0.02 7.62 ± 3.595 The authors are highly thankful to Department of Science and
FF12 0.20 ± 0.02 1.90 ± 0.824 Technology, Fund for Improvement of Science and Technology
FF13 0.09 ± 0.02 7.62 ± 0.824 Infrastructure (FIST) (Grant No.: SR/FST/LSI – 607/2014),
FF14 0.06 ± 0.02 26.19 ± 5.016
FF15 0.09 ± 0.01 18.57 ± 4.285
Government of India, for providing equipment facility. The authors
FF16 0.06 ± 0.02 70.95 ± 4.285 would like to acknowledge Evonik, Germany and Jawa
Pharmaceuticals, Gurgaon, India for providing gift samples of
PLGA and GS, respectively. The authors also thank Indian
89.98 ± 0.15% and 90.1 ± 0.01% for PBS pH 6.4 and SWF pH 7.4 at Pharmacopoeia Commission, Ministry of Health and Family
216 h (9th day). Drug release was found to follow Higuchi Matrix Welfare, Ghaziabad, India for providing DSC, FT-IR and Water by
model and Korsemeyer–Peppas model for batch F2 in PBS pH 6.4 Karl Fisher facility.
and SWF pH 7.4, respectively. Antimicrobial effectiveness study
confirmed that PNPs were effective against SA and PA. XRD con-
Disclosure statement
firmed amorphous nature of polymer being retained in the PNPs
as well as film and also entrapment of GS in PNPs. The prepared The authors report no declarations of interest.
PNPs incorporated in fast dissolving film may reduce the fre-
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