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-

negative infections [13].
Wound healing involves interactions at cellular and molecular
Conventional dosage forms have limitations due to high fre-
level in a well-defined manner. The steps include inflammation,
quency of administration and development of antimicrobial resist-
cell migration, cell proliferation and tissue remodeling [1]. The
ance. The alternative to the same is local therapy in the form of
duration of these phases may vary from few weeks to months
antiseptic impregnated drapes, antimicrobial coated gauge, and
depending upon the type of wound [2,3]. Surgical site infection
antibiotic creams. Several researchers have used collagen-based
(SSI) is a pathological condition observed after surgery. In the GS implant/sponge (GCIS) after surgical procedures. Almedia et al.
patients with SSI, prolonged inflammatory phase is observed due reported no SSI in patients receiving GCIS compared to control
to cellular shock subsequent to incision. SSI is associated with group [14]. Brehant et al. through research finding proved that
microbial growth and biofilm formation, with more than 1000 col- application of GCIS reduced SSI in patients upto 9% after colorec-
ony-forming units at the site of infection [4,5]. Global researches tal surgery [15]. Benedetto et al. found that out of 329 patients
have shown that some of the microbial strain coexisting with receiving GCIS only 0.6% patients showed the deep sternal wound
highest probability are Staphylococcus aureus (SA), Pseudomonas infection (DSWI) and out of the rest; 2.01% patients showed symp-
aeruginosa (PA), Escherichia coli and other associated strains toms of DSWI [16]. It was observed and reported that delivering
include Corneybacterium, Peptoniphilis, Serratia marcences, GS prophylactically after surgical procedure at the surgical site sig-
Anaerococcus sp, Finegolida magna, Coagulase negative strepto- nificantly reduces the number of patients reported with
cocci, Methicillin resistant S. aureus (MRSA) [6–11]. SSI [17–20].
As per Centers for Disease Control and Prevention guidelines, Poly lactic-co-glycolic acid (PLGA) has been approved by
the management of SSI involves pre-operative, intra-operative, United States Food and Drug Administration as biodegradable
and postoperative management. Gentamicin sulfate (GS) is and biocompatible polymer with wide range of erosion times.
injected prophylactically to maintain therapeutic levels of drug These properties makes it suitable controlled release polymer
during surgical procedures [12]. Oral consumption of aminoglyco- [21–23]. Several researchers have successfully prepared PLGA poly-
sides, such as GS and vancomycin, is preferred alternative for pre- meric nanoparticles (PNPs) of water soluble and insoluble drugs
venting SSI after surgery. GS has been approved by WHO as an 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- Nanoparticle fabrication
trolled release property. Chitosan and/or alginate PNPs require PNPs were prepared by water-in-oil-in-water (w/o/w) double emul-
chemical modification to provide controlled release of drug. The sion solvent removal method. Briefly, 50 mg/ml aqueous solution
maximum duration of drug release achieved with chemically of GS was prepared in double-distilled water (DDW). PLGA, in dif-
modified PNPs containing chitosan and alginate is less (upto ferent GS to PLGA ratio was dissolved in fixed volume of ethyl
6 days) compared to PLGA [24–28]. Thus, PLGA was selected as acetate with the help of a mechanical stirrer (2 MLH, Remi labora-
polymer for the preparation of PNPs. Gum Kondagogu (GKK), an tory Instruments, Mumbai, India). Primary emulsion was prepared
exudate from the bark of Cochlospermum gossipium is a nontoxic by ultrasonicating the organic phase with an aqueous phase in
polysaccharide. Chemically, GKK is composed of arabinose, galact- the presence of PVA as an emulsifier using Vibra Cell probe soni-
ose, glucose, mannose, rhamnose, glucuronic acid, and galactour- cator (Sonics and Materials Inc., CT) for 2 min. The primary emul-
onic acid [29–31]. The emulsification property of GKK for the sion was re-emulsified in GKK solution followed by particle
preparation of liquid paraffin emulsion was evaluated between hardening and ethyl acetate removal. The PNPs were collected by
concentrations 0.1% and 0.6% [32]. centrifugation at 10,000 rotation centrifugal force (rcf) for 20 min
The objective of present investigation was development of GS- using cooling centrifuge (Remi Pvt. Ltd., India). PNPs were washed
loaded PLGA-based PNPs coated with GKK for the treatment of with DDW and lyophilized using lyophilizer (Delvac, Tamilnadu,
SSI. Screening study using Plackett–Burman design and Design of India) for storage and further studies. Lyophilization can increase
Experiment (DoE) study using Box–Behnken design were used for long-term storage of polymeric nanoparticles [34]. The lyophilized
the fabrication of PNPs. The PNPs were loaded in pullulan-based PNPs were stored in microcentrifuge tubes sealed with parafilm
film. Semisolid dosage forms, such as creams and gels, have limi- at 20
C.
tation of uneasiness and discomfort at the site, messiness, and Factors involved in the fabrication of PNPs were screened by
spread. Thus, film was selected due to convenience and accept- Plackett–Burman design using Minitab (Version 17, Minitab Inc.,
ability as compared to conventional forms for application at the USA). Based on the results of the Plackett–Burman design, three
surgical site during closure of the surgical incision. The PNPs and significant factors were identified. Each factor was considered at
film were evaluated for various parameters such as estimation of two levels before applying DoE study using Box–Behnken design.
particle size and polydispersity index (PDI), zeta potential, surface
morphological estimation, in vitro drug dissolution study, anti-
Preparation of films
microbial effectiveness study, residual solvent analysis, in vitro dis-
Film was prepared by solvent casting method. Trial batches
integration study of film, drug content in film, estimation of
included formulation of pullulan-based film at 2% and 5% w/v
mechanical properties of film and X-ray diffraction study (XRD).
concentration. To an aqueous solution of pullulan, sufficient
amount of plasticizer polyethylene glycol 400 (PEG 400) or propyl-
Material and methods ene glycol (PG) were added individally. Before pouring the solu-
tion into teflon petri-plate, the solution was stirred using
Materials magnetic stirrer (Remi, Delhi, India) for 1 h at 100 rpm. Films were
Gift sample of GS, PLGA 50:50 (Resomer RG 502) having molecular dried at 40
C in hot air oven (EIE instruments Pvt. Ltd.,
weight 7 to 17 KDa and GKK were received from Jawa Ahmedabad, India) for 36 h. PNPs containing films were prepared
Pharmaceuticals (Gurgaon, India), Evonik Industries (Evonik by dissolving required amount of pullulan and plasticizer in the
Industries, Essen, Germany) and Nutrioma Healthcare (Hyderabad, aqueous solution of PNPs. The resulting dispersion was stirred for
India), respectively. Sodium chloride, potassium chloride, sodium 1 h at 100 rpm. The solution casting and drying conditions were
hydrogen carbonate, di-sodium hydrogen orthophosphate, potas- same as described previously.
sium dihydrogen orthophosphate, sodium di-hydrogen orthophos-
phate, polyvinyl alcohol (PVA) having molecular weight 85 to
Evaluation of PNPs and film
124 kDa were purchased from CDH Laboratories Pvt. Ltd. (New
Percentage yield (PY). The PY was calculated as the percentage of
Delhi, India). Nutrient agar media, nutrient broth and cellulose-
the weight of prepared PNPs to the total amount of components
based dialysis membrane LA401 with molecular weight cutoff
(GS, PLGA, PVA and GKK) used in the process as shown in
12–14 KDa were procured from Himedia Laboratories Pvt. Ltd.
Equation (1).
(Mumbai, India). SA (Microbial Type Culture Collection (MTCC
6538) and PA (MTCC 9047) were procured from Council for Weight of PNPs prepared
PY ¼  100 (1)
Scientific and Industrial Research (CSIR) - Institute of Microbial Total weight of components
Technology (Chandigarh, India).
Mean particle size and PDI. The mean particle size (d.nm) and PDI
Methods were measured using four-side open disposable cuvette and zeta
potential (mV) was measured using zeta potential measuring cell
Physical compatibility study using particle size analyzer (Horiba India Pvt. Ltd, Bangalore) in
During pharmaceutical processing or storage, physical interaction duplicate involving dynamic light scattering (DLS) technique.
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 Evaluation of surface morphology
1:1 in Biological Oxygen Demand (BOD) incubator (MOSIV – 3, The morphological evaluation of the lyophilized PNPs was carried
Sonar, New Delhi, India) at 40
C for 21 days [33–34]. The sample out by scanning electron microscopy (SEM) (Carl Zeiss Evo 18 SEM
were then analyzed using differential scanning calorimetry (DSC) with EDXRF, Jena, Germany) and transmission electron microscopy
(DSC-6000, Perkin Elmer, MA). (TEM) (FEI Tecnai 20 Hillsboro, USA). For SEM, the PNPs were
 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 807

coated with gold layer which imparts sufficient conductivity to the sodium hydrogen carbonate and 0.35 g sodium di-hydrogen phos-
particles before being examined. Double side adhesive carbon phate in 100 ml DDW, adjusted to pH 7.4 [40]. Prior to the study,
tape was placed on the sample mount. Gold-layered PNPs were the dialysis bag was treated as per the procedure specified by
placed on the upper side of carbon tape and sample mount was Himedia [41] and stored in dissolution media.
placed in the holder to capture SEM micrographs. For TEM, PNPs After centrifugation, the supernatant was separated and PNPs
were dispersed in DDW and placed on the coated copper grids in the form of viscous liquid were collected in a microcentrifuge
with mesh size 60 and allowed to dry in air. The copper grid was tube. From the microcentrifuge tube, 2 ml PNPs containing viscous
placed on the grid holes, locked with locking flange in the piezo liquid was transferred into the dialysis bag, tied and suspended in
holder and TEM images were recorded. 200 ml dissolution medium maintained at 37
C. Dissolution
medium equivalent to 2 ml was withdrawn at predetermined time
Drug loading (DL) and entrapment efficiency (EE) in PNPs. DL is 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,
defined as the amount of drug to amount of lyophilized PNPs pre- 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
pared and EE is defined as the amount of drug entrapped in
of GS was performed in duplicate by the method described earlier
nanoparticles over total amount of drug taken.
for the estimation of DL at 335 nm.
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, Antimicrobial effectiveness study. Identification of the zone of
India). The aqueous layer of the solution was used to estimate the inhibition (ZOI) with PNPs was carried out as per procedures
content of GS in duplicate. specified in Indian Pharmacopoeia for antimicrobial assay with
After centrifugation process, the pellet and centrifuged solu- slight modification [38]. GS equivalent to 25 mg/ml concentration
tion were separated. This centrifuged solution here is referred to was prepared from stock solution of PNPs and marked as S3 (S3
as supernatant. The pellet was washed with DDW and centri- represents the median level concentration). Other dilutions con-
fuged again. The resulting liquid after re-centrifugation is taining GS equivalent to 16, 25, 31.20 and 39.05 mg/ml were pre-
referred as wash medium. The two solutions were combined and pared from stock solution and marked as S1, S2, S4 and S5,
used for the estimation of EE. The estimation was performed respectively. The concentrations were selected such that the ratio
in duplicate. between each lower and higher level remained 4:5. Nutrient agar
The amount of GS was estimated by validated derivatization plates containing microbial culture (1 ml) were prepared and 4
method suggested by Kowalczuk et al. with minor modifications wells were made in each petri-plate using 0.6-mm stainless steel
[35]. Calibration curve for GS was plotted from 1.6 mg/ml to borer. Wells were filled with 0.5 ml PNPs solution in a manner that
25.6 mg/ml concentration and the value of correlation coefficient each alternate well in every petri-plate received S3 and other wells
(R2) was found to be 0.99. Derivatization reagent was prepared by received S1, S2, S4, and S5, respectively, in duplicates. The ZOI
was measured in the petri-plates after an incubation for 24 h at
dissolving 20 mg of o-phthalaldehyde (OPA) in 1.0 ml methanol
37
C in BOD incubator (Samiksha Industrial Corporation, Mumbai,
followed by addition of 0.1 ml thioglycolic acid and diluting the
India). A graph between square of ZOI versus log concentration
solution to 10 ml with 0.20 M boric acid solution adjusted to pH
was plotted and MIC was estimated by taking antilog of the con-
10 with 40% sodium hydroxide solution. 2.50 mM hydroxy propyl
centration obtained by extrapolating the curve till it intersected
– b – cyclodextrin solution was used as reaction stabilizer. The GS-
the x-axis.
OPA complex was prepared by reacting a mixture containing
For the measurement of MIC using nutrient broth, a stock solu-
0.50 ml sample solution, 0.50 ml derivatization reagent and 1.50 ml
tion of GS and GS equivalent in PNPs was prepared by dissolving
stabilizer at 60
C for 15 min followed by cooling to room tem-
sufficient weight in DDW to obtain a concentration of 1000 mg/ml
perature and estimated using UV-Visible spectrophotometer
for GS and 300 mg/ml for GS equivalent in PNPs. From the stock
(Jasco, Mumbai, India) at 335 nm. Percentage EE (% EE) and per-
solution, sufficient volume was pipetted in different test tubes
centage DL (% DL) were calculated by Equations (2 and 3) [36].
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
Total drug ðmgÞFree drug ðmgÞ
% EE ¼  100 (2) 1.92 to 61.44 mg/ml for GS and GS equivalent in PNPs, respectively.
Total drug ðmgÞ The test tubes were incubated for 24 h at 37
C in BOD incubator
and evaluated for the presence of growth.
Total drug ðmgÞFree drug ðmgÞ
% DL ¼  100 (3)
Total weight of PNPs ðmgÞ
Estimation of residual solvents in PNPs. Ethyl acetate was used as
one of the components during fabrication of PNP. It is a class III
In vitro dissolution study. In vitro dissolution study was performed solvent having acceptable limit below 5000 mg/ml as per ICH Q3C
using dialysis bag (LA 401, Himedia, Mumbai, India) in two differ- (R5) guideline [42]. The level of ethyl acetate was estimated by
ent dissolution media, namely phosphate buffer saline (PBS) pH gas chromatographic analysis performed on Agilent Gas chro-
6.4 and simulated wound fluid (SWF) pH 7.4. During surgical pro- matograph equipped with head space (GC-HS) (Agilent
cedure, incision disrupts skin pH and shifts it to pH above 6 pro- Technologies 7890 A, New Delhi, India) using Agilent capillary col-
viding rationale for the selection of PBS pH 6.4 as dissolution umn, DB-624, with dimensions 30 m  0.25 mm  1.4 mm, Nitrogen
medium [37]. PBS pH 6.4 was prepared as per method specified in as carrier gas maintained at a flow rate of 0.50 ml/min with 500 ml
Indian Pharmacopoeia 2010 by dissolving 1.79 g of disodium as injection volume. The run time was kept constant for 45 min.
hydrogen orthophosphate, 1.36 g of potassium dihydrogen ortho- Sufficient amount of lyophilized powder of PNPs was dispersed
phosphate and 7.02 g of sodium chloride in 1000 ml of DDW [38]. in carrier solvent, NN0 -dimethyl formamide and injected in tripli-
Chronic wounds exist at pH range of 7.2 to 8.9 providing rationale cate. The chromatogram was compared with standard chromato-
for selection of SWF pH 7.4 [39]. SWF pH 7.4 was prepared by dis- gram of ethyl acetate. Concentration of ethyl acetate in PNPs was
solving 0.68 g sodium chloride, 0.22 g potassium chloride, 2.5 g 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 Level () Level (þ)
A Type of PLGA (Categorical) End capped Uncapped
B Volume of Ethyl acetate (ml) 5 15
C Quantity of PLGA(mg) 75 (1:1.5) 225 (1:4.5)
D Concentration of Primary emulsifier (%) 0.5 3.5
E Duration of Sonication (min) 2 5
F Temperature while sonication (Categorical) Ice Bath RT
G Concentration of Secondary emulsifier (%) 0.05 0.35
H Volume of secondary emulsifier (ml) 20 40
I Speed of stirring (rpm) 200 800
J Duration of stirring (h) 2 8

Concentration of organic solvent ðlg=mlÞ Results and discussion

Area of standard solvent Dilution of test
¼  (4) Nanoparticle fabrication and evaluation
Area of test solvent Dilution of standard
 Potency of standard  1000 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].
In vitro disintegration study. It was performed by placing Water-soluble drugs tends to partition between solvents therefore
1  1 cm2 film in a petri-plate containing 10 ml SWF. The time double emulsion solvent removal technique was adopted for the
taken for the disintegration of film was measured in duplicate and fabrication of PNPs. Numerous process-related factors having
considered as in vitro disintegration time. effect on DL including drug-to-polymer ratio, volume of solvent,
concentration of primary and secondary emulsifier, pH and ionic
Drug content in film. A portion of film having dimension 2  1 cm2 strength of outer medium were considered. Several trial batches
was cut, weighed, and dissolved in 1 ml DDW and vortexed with were prepared prior to Plackett–Burman design. PLGA was solubi-
an equal volume of ethyl acetate. From the vortexed solution, lized in two different organic solvents (ethyl acetate and dichloro-
0.50 ml of aqueous medium was used for the estimation of GS in methane), PVA was used at variable concentration between 2%
film. The drug content was measured in duplicate as per the and 3.5% as primary emulsifier and emulsification was done by
method described in the section of measurement of % DL. 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
Mechanical properties of film. Mechanical properties of film
unaltered pH of GKK solution), and addition of salt
namely tensile strength (TS) and % elongation (% E) were esti-
were considered.
mated in duplicate using Digital Tensiometer (EIE Instruments,
Ethyl acetate is partially miscible with water and molecular dis-
Ahmedabad, India). Film with dimensions 1.5  7 cm2 free from
persion between ethyl acetate and PLGA yields higher DL [44,45].
physical imperfection were cut and estimated for thickness using
PNPs prepared with dichloromethane had higher DL and smaller
Digital Thickness gauge (Mitutoyo Corporation, Kanagawa, Japan).
particle size in comparison to PNPs prepared using ethyl acetate.
The film was held between two clamps of digital tensiometer.
PLGA possess glass transition close to 50
C. Heat generation was
One of the clamps was stationary and other clamp moved down
observed during ultrasonication process. Lower DL was observed
to pull the film at rate of 35 inch/min. The clamps were positioned
with batches prepared at high amplitudes. Lower DL was seen in
at a distance of 3 cm. Equations (5 and 6) were used to calculate
the PNPs prepared after adding salt to the solution of GKK. Acidic
TS and % E, respectively.
pH showed lower DL, however lower PY was observed with basic

Force required to break film ðkgÞ pH. Unaltered pH of GKK solution was further selected. Based on
TS N=cm2 ¼  9:8
Initial cross sectional area of the film ðcm2 Þ these observations, Plackett Burman design was applied for
(5) screening of 10 significant factors (2 categorical and 8 numerical)
Increase in length ðmmÞ each considered at 2 levels as summarized in Table 1.
% E¼  100 (6)
Initial length of the film ðmmÞ 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.
XRD of film and PNPs. XRD of GS, PLGA, GKK, optimized batch of Although the usual PDI range is between 0 to 1; however, PDI
PNPs and optimized batch of film FF16 were performed using more than 1 was observed with batches FP1, FP3-FP5, FP7-FP8,
Xpert MPD X-ray diffractometer (Philips, Holland) at 45 kV and and FP10-FP12 indicated particles of improper shape. Thus, condi-
30 mA. It uses copper Ka X-ray radiation source and graphite tion for preparation of these batches were not acceptable.
monochromator to produce X-rays of 1.5405 Ð. Sample was Batches FP1 and FP3 were found to have lower DL (<20%), higher
placed in copper holder, placed above the sample tray and ana- PDI (>9.0) and lower particle size (30 nm). Batches FP4, FP8,
lyzed between 5
to 50
. 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
Statistical analysis
size and PDI. It was also seen with these batches that smaller par-
Statistical software, Minitab was used for screening of factors ticles possessed higher % DL. Acceptable PDI and particle size was
using Plackett–Burman design and DoE study using Box–Behnken observed with batches FP2, FP6, and FP9. Average particle size
design. The results were considered statistically significant when was close to 250 nm with PDI between 0.50 and 0.70. Level of sig-
the obtained p values was <0.05. 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 However, higher concentration of GKK favored DL and particle
Minitab software for % EE, % DL, particle size, and PDI, respect- size with lower % EE and higher PDI. Volume of GKK solution did
ively, as shown in Figure 1 (A–D). Comparing the effects of all fac- not show much change on the response parameters. Speed and
tors, factors B, D, and G were found significant (p < 0.05). duration of stirring showed similar responses, that is, increasing
Main effects plots suggest the effect of different factors on the speed and duration yields PNPs with higher % EE, % DL, par-
PNPs. Type of PLGA showed an effect on PDI, whereas % DL, % ticle size, and lower PDI.
EE, and particle size remained unchanged. Volume of ethyl acetate Further, DoE study using Box–Behnken design was performed
used to solubilize PLGA had a drastic effect on % DL, particle size, for three factors, namely volume of ethyl acetate, concentration of
and PDI. Lower volume of ethyl acetate was found beneficial for primary emulsifier, and concentration of secondary emulsifier each
PNPs fabrication. Higher amount of PLGA for PNPs preparation considered at two levels. As summarized in Table 3, 15 batches
yielded higher % DL, lower PDI, whereas % EE and particle size FB1 to FB15 were prepared.
were less affected. With higher concentration of PVA, % DL, % EE, All the prepared batches had particle size ranging between
and PDI increased and particle size decreased. Duration of sonic- 214 nm to 348 nm and PDI above 0.20. Batches FB1, FB3, FB6, FB7,
ation did not show much effect on the PNPs. Ice bath yielded FB8, FB13, and FB15 showed % DL above 30%, whereas batches
PNPs with higher % DL, % EE and particle size and lower PDI. 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

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-
Figure 4. Composite desirability and optimization condition obtained using pended in a solvent, there is a tendency of adherence of thin
Box–Behnken design. electric dipole layer of the solvent over the particles [49].

% DL and in vitro dissolution study

Physical compatibility study
The in vitro dissolution study was performed for two batches, F1
Figure 5(A) shows the DSC thermograms for PLGA, GS, and phys- and F2. F1 indicates batch selected from design space generated
ical mixture. The thermogram of PLGA (Thermogram A) illustrates with Minitab software, whereas F2 refers to the optimized batch.
glass transition temperature (Tg) at 37
C confirming its amorph- The composition of batches F1 and F2 are summarized in Table 4.
ous and glassy nature with rigid chain structure [22,46]. Melting Before transferring the PNPs in the form of viscous liquid into the
was observed at peak temperature 50.46
C for PLGA. dialysis bag, amount of GS was estimated. %DL for the batches F1
Thermogram B exemplifies DSC of GS and two endothermic peaks and F2 was found to be 48 and 60%w/w, respectively.
were observed. Similar to the description given by Rosenkrantz Figure 5(D) shows the cumulative percentage drug release for
et al. broad endothermic peak from 47.11
C to 140.01
C was batches F1 and F2 up to 216 h in PBS pH 6.4 and SWF pH 7.4.
observed with its peak at 90.60
C due to loss of water. A sharp Sustained release of GS was observed from PNPs. The release
endothermic peak at 244.13
C corresponding to melting decom- from PNPs occurs due to hydrolytic cleavage of the linkage
position of gentamicin was also observed [47]. Thermogram C between lactic acid and glycolic acid in PLGA mediated through
embodies 1:1 physical mixture of GS and PLGA. Intact peaks for steps such as diffusion, solvent penetration, degradation and ero-
PLGA and GS were observed at 47.3
C and 247.37
C, respectively, sion [50]. PLGA is known to show initial burst release [22].
which confirm the physical compatibility between them. 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
Percentage yield and particle size estimation 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
The PY for the F2 batch of PNPs was 77.68 ± 5.60%. The mean par- pH 7.4, respectively. However, similar drug release of 83.7 ± 0.10%
ticle size estimated by DLS technique was found to be and 84.4 ± 0.06% was observed at 216 h using PBS pH 6.4 and
280 ± 12.04 nm. Based on the PDI value 0.15 ± 0.01, it was con- SWF pH 7.4, respectively. Similar patterns of drug release for batch
firmed that the PNPs were monodispersed. A 0.20% w/v aqueous F2 in PBS pH 6.4 and SWF pH 7.4 were also observed. Drug
solution of GKK used as secondary emulsifier possessed release for batch F2 at 1 h, 6 h and 24 h was 11.85 ± 0.01%,
42.4 ± 1.64 mV charge. Naidu et al. demonstrated that GKK pos- 22.66 ± 0.36%, and 32.46 ± 0.22% using PBS pH 6.4 and
sess high negative zeta potential at low concentration which may 30.6 ± 0.4%, 34.3 ± 0.27%, and 48.4 ± 0.32% using SWF pH 7.4,
be attributed due to the presence of large number of carboxylic respectively. Drug release of 89.98 ± 0.15% and 90.1 ± 0.01% was
acid and hydroxyl groups [48]. The zeta potential possessed by observed at 216 h using PBS pH 6.4 and SWF pH 7.4, respectively.
PNPs was found to be 4.9 ± 0.84 mV. The difference in the result- Both batches F1 and F2 showed initial drug release followed by
ant potential after electrostatic interaction is approximately gradual controlled release up to 216 h indicating suitability for SSI
>þ35 mV, it confirms the stability of the solute particles of pri- treatment. Higher drug release for batches F1 and F2 was
mary emulsion. observed using SWF pH 7.4 compared to PBS pH 6.4. The reason
 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 813

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 and 2.11 for batch F1 and 6.96 and 2.11 for batch F2 respectively
could be due to property of PLGA. PLGA is more likely to show was obtained. As the t-stat value > t-critical value, it was con-
less pronounced degradation at slight acidic pH and alkaline cluded that pH of dissolution medium significantly affected drug
media is favorable for degradation [22]. To confirm the effect of release. The dissolution study data was also statistically analyzed
pH of dissolution medium on dissolution profiles, t-test was using DD solver (Microsoft office 2013) for the determination of
applied on the results of dissolution study for batches F1 and F2. dissolution models [51]. The drug release order was selected on
The values of t-stat and t-critical were generated by using the basis of regression coefficient (r2) obtained by plotting graphs
Microsoft excel 2013. The t-stat value and t-critical value of 6.72 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 agar plate method, MIC for PNPs containing GS was found to be
model with r2 value 0.94 and 0.98 for batches F1 and F2, respectively, 6.84 mg/ml and 13.07 mg/ml for SA and PA, respectively. Table 5
whereas PNPs in SWF pH 7.4 followed Korsmeyer–Peppas model summarizes the result of antimicrobial study against SA and PA
with r2 value 0.97 and 0.96 for batches F1 and F2, respectively. In for different concentration of GS in free state and PNPs containing
Higuchi matrix model, the drug release depends on the concentra- GS. GS at concentration 15.36 mg/ml and 30.72 mg/ml showed
tion gradient of the solute molecules, whereas Korsmeyer–peppas inhibitory activity against SA and PA, respectively. The effective
model clearly signifies release from polymeric systems [52]. Further, concentration of GS in free state and PNPs containing GS were
the diffusion kinetics was estimated using Equation (8). found to be similar for PA; however, PNPs containing GS showed
inhibition in growth of SA at much lower concentration compared
slope of the curve obtained with korsemeyer peppas model
n¼ to GS in free state. The difference in effectiveness may be attrib-
2:303
uted to the surface charge of PNPs [53].
(8)
where n represents a numerical factor correlated to type of diffu- Estimation of residual solvents in the PNPs
sion followed. If the value of n lies between 0 to 0.5, the release
Figure 7(A) shows standard solution of ethyl acetate has a reten-
is fickian, if the value of n lies between 0.5 to 1, the diffusion is tion time at 14.32 min. Figure 7(B) shows chromatogram for PNPs
considered as nonfickian. The value for ‘n’ for the batches F1 and a peak at retention time 14.34 min indicating the presence of
F2, was found to be 0.23 and 0.27; 0.13, and 0.12 in dissolution ethyl acetate in the PNPs. The amount of ethyl acetate was found
media PBS pH 6.4 and SWF pH 7.4, respectively indicating fick- out to be 489 ± 0.28 mg/ml in the PNPs which is below the accept-
ian diffusion. able limit, 5000 mg/ml as per ICH Q3C (R5) guidelines for residual
solvents [42]. The observed amount endorsed acceptable limit of
Antimicrobial effectiveness study ethyl acetate as residual solvent in PNPs.
Figure 6(A) depicts the plot of log dose (log concentration) versus
Preparation of film
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 Films using pullulan were prepared at varying concentration as
PA. From the observations of antimicrobial study performed using 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

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
Figure 7. Chromatograms obtained from GC-HS analysis (A) Standard and as optimized batch due to highest % E and acceptable TS.
(B) PNPs.

respectively, were stiff and brittle. These batches had an average X-ray diffraction study
thickness 0.17 ± 0.01 mm and 0.26 ± 0.03 mm. Batch FF3 to FF10
Figure 8 shows X-ray diffraction pattern for GS, PLGA, pullulan,
containing plasticizer PEG 400 or PG were flexible, smooth with
GKK, optimized batch of PNPs, and optimized batch of film FF16.
better mechanical properties. The average thickness for the films
Figure 8(A) depicts single sharp peak at 44.61
for GS. The pres-
prepared with PG as plasticizer varied between 0.07 ± 0.03 mm to
ence of single sharp peak may be due to the recrystallization of
0.17 ± 0.08 mm, whereas films prepared with PEG 400 as plasticizer
GS. Figure 8(B–D) shows no sharp diffraction peak, and thus, it
possessed thickness between 0.17 ± 0.02 to 0.24 ± 0.05 mm. Films was established that PLGA, pullulan, and GKK exist in amorphous
containing PG had lower thickness, thus PG was selected for the form. Diffractogram of the optimized batch of PNPs indicated
preparation of PNPs loaded film FF13–FF16. The thickness of PNPs sharp peaks at 9.57
, 20.25
, 25.01
, 35.95
, 40.18
, 43.35
and
incorporated batches FF13 and FF14 were found to be 44.95
(Figure 8(E)). The presence of peak at 44.95
indicated sur-
0.15 ± 0.02 mm and 0.18 ± 0.06 mm, whereas the thickness of the face associated GS in PNPs confirming loading of GS. No specific
batches FF15 and FF16 was found to be between 0.27 ± 0.07 mm peak was observed for optimized batch of film FF16 (Figure 8(F))
and 0.24 ± 0.04 mm, respectively. Batches FF12, FF15, and FF16 indicating its amorphous surface and incorporation of PNPs inside
had nearly 50% DL. Maximum % DL was observed with batch the film and not over the surface of the film.
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). Conclusion
Batches FF1 to FF10 prepared without PNPs showed rapid in
GS-loaded PLGA-GKK encapsulated PNPs were formulated for the
vitro disintegration time of less than 1 min in SWF. PNPs contain-
treatment of SSI at localized site. Controlled release of GS was
ing batches (FF11 to FF16) showed longer in vitro disintegration
observed due to PLGA after an initial burst release. The burst
time. Batches FF15 and FF16 showed maximum in vitro disintegra- release is effective for the attainment of initial concentration of
tion time of 6 min and 8 min, respectively this might be due to GS at site of application. Batches F1 and F2 showed initial drug
the higher concentration of pullulan. Higher in vitro disintegration release followed by controlled release up to 216 h indicating suit-
time was desirable as it provided time for ease of application at ability for SSI treatment. Drug release was affected by pH of the
the surgical site. Thus, batches FF15 and FF16 were selected. 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
Estimation of mechanical properties of film
optimization. PNPs prepared with optimized batch had particle
Batches FF11 to FF16 were evaluated for mechanical properties size less than 280 nm and PDI close to 0.15 indicating monodis-
using Digital Tensiometer. The observations are summarized in perse nature of the system. The PNPs possessed spherical and
Table 7. It was observed that, TS decreased and %E increased as smooth surface as confirmed by SEM and TEM study. Batch F2 of
amount of PG increased. As concentration of pullulan increased, 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-
quency of administration and increase the availability of GS at the References
surgical site.
[1] Singer AJ, Clark RAF. Cutaneous wound healing. N Engl J
Med. 1999;341:738–746.
Acknowledgements
[2] Alba-Loureiro TC, Hirabara SM, Mendonca¸ JR, et al.
The authors would like to acknowledge Institute of Pharmacy, Diabetes causes marked changes in function and metabol-
Nirma University, Ahmedabad, India for providing the facility and ism of rat neutrophils. J Endocrinol. 2006;188:295–303.
 DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 817

[3] Beer HD, Longaker MT, Werner S. Reduced expression of [22] Makadia HK, Siegel SJ. Poly lactic-co-glycolic acid (PLGA) as
PDGF and PDGF receptors during impaired wound healing. biodegradable controlled drug delivery carrier. Polymers
J Invest Dermatol. 1997;109:132–138. (Basel). 2011;3:1377–1397.
[4] Donlan RM. Biofilms: Microbial life on surfaces. Emerging [23] Bao X, Gao M, Xu H, et al. A novel oleanolic acid-loaded
Infect Dis. 2002;8:881–890. PLGA-TPGS nanoparticle for liver cancer treatment. Drug
[5] Wolcott RD, Gontcharova V, Sun Y, et al. Bacterial diversity Dev Ind Pharm. 2015;41:1193–1203.
in surgical site infections: not just aerobic cocci any more. J [24] Sun L, Chen Y, Zhou Y, et al. Preparation of 5-fluorouracil-
Wound Care. 2009;18:317–323. loaded chitosan nanoparticles and study of the sustained
[6] Giacometti A, Cirioni O, Schimizzi AM, et al. Epidemiology release in vitro and in vivo. Asian J Pharm Sci. 2017;12:
and microbiology of surgical wound infections. J Clin 418–423.
Microbiol. 2000;38:918–922. [25] Motiei M, Kashanian S. Preparation of amphiphilic chitosan
[7] Cronquist AB, Jakob K, Lai L, et al. Relationship between nanoparticles for controlled release of hydrophobic drugs.
skin microbial counts and surgical site infection after neuro-
J Nanosci Nanotechnol. 2017;17:5226–5232.
surgery. Clin Infect Dis. 2001;33:1302–1308.
[26] Azevedo MA, Bourbon AI, Vicente AA, et al. Alginate/chito-
[8] Cantlon CA, Stemper ME, Schwan WR, et al. Significant
san nanoparticles for encapsulation and controlled release
pathogens isolated from surgical site infections at a com-
of vitamin B2. Int J Biol Macromol. 2014;71:141–146.
munity hospital in the Midwest. Am J Infect Control. 2006;
[27] Souza JM, Caldas AL, Tohidi SD, et al. Properties and con-
34:526–529.
trolled release of chitosan microencapsulated limonene oil.
[9] Edmiston CE, Krepel CJ, Seabrook GR, et al. Anaerobic infec-
tions in the surgical patient: microbial etiology and therapy. Brazilian J. Pharmacogn. 2014;24:691–698.
Clin Infect Dis. 2002;35:S112–S118. [28] Di Martino A, Kucharczyk P, Capakova Z, et al. Chitosan-
[10] Shahane V, Bhawal S, Lele U. Surgical site infections: A one based nanocomplexes for simultaneous loading, burst
year prospective study in a tertiary care center. Int J Health reduction and controlled release of doxorubicin and 5-fluo-
Sci (Qassim). 2012;6:79–84. rouracil. Int J Biol Macromol. 2017;102:613–624.
[11] Patel Sachin M, Patel Mitesh H, Patel Sangeeta D, et al. [29] Vinod VTP, Sashidhar RB. Solution and conformational
Surgical site infections: Incidence and Risk Factors in a properties of gum kondagogu (Cochlospermum gossypium)
Tertiary Care Hospital, Western INDIA. Natl J Community – a natural product with immense potential as a food addi-
Med. 2012;3:193–196. tive. Food Chem. 2009;116:686–692.
[12] Burke JF. The effective period of preventive antibiotic [30] Vinod VTP, Sashidhar RB, Suresh KI, et al. Morphological,
action in experimental incisions and dermal lesions. physico-chemical and structural characterization of gum
Surgery. 1961;50:161–168. kondagogu (Cochlospermum gossypium): a tree gum from
[13] 19th WHO Model list of Essential Medicines. Geneva: World India. Food Hydrocoll. 2008;22:899–915.
Health Organization. 2015;19:1–45. [31] Vinod VTP, Sashidhar RB, Sarma VUM, et al. Compositional
[14] Costa Almeida CEP, Reis L, Carvalho L, et al. Collagen Analysis and Rheological Properties of Gum Kondagogu
implant with gentamicin sulphate reduces surgical site (Cochlospermum gossypium): a Tree Gum from India.
infection in vascular surgery: a prospective cohort study. Int J Agric Food Chem. 2008;56:2199–2207.
J Surg. 2014;12:1100–1104. [32] Naidu Vegi GM, Sistla R, Srinivasan P, et al. Emulsifying
[15] Brehant O, Sabbagh C, Lehert P, et al. The gentamicin-colla- properties of gum kondagogu (cochlospermum
gen sponge for surgical site infection prophylaxis in colo- gossypium), a natural biopolymer. J Sci Food Agric. 2009;
rectal surgery: a prospective case-matched study of 606 89:1271–1276.
cases. Int J Colorectal Dis. 2013;28:119–125. [33] Van Dooren AA, Duphar BV. Design for drug-excipient
[16] Benedetto U, Raja SG. Scoring system to guide decision interaction study. Drug Delvelopment Ind Pharm. 1983;9:
making for the use of gentamicin-impregnated collagen
43–55.
sponge to prevent deep sternal wound infection. J Thorac [34] Fonte P, Reis S, Sarmento B. Facts and evidences on the
Cardiovasc Surg. 2014;148:2390–2396.
lyophilization of polymeric nanoparticles for drug delivery.
[17] Raja SG. Local application of gentamicin-containing colla-
J Control Release. 2016;225:75–86.
gen implant in the prophylaxis and treatment of surgical
[35] Kowalczuk D, Pietras R, Paw B, et al. Applying liquid chro-
site infection following cardiac surgery. Int J Surg. 2012;10:
matography with fluorescence detection to determine gen-
S10–S14.
[18] Knaepler H. Local application of gentamicin-containing col- tamicin. Polish J Environ Stud. 2010;19:587–591.
[36] Tran BN, Nguyen HT, Kim JO, et al. Developing combination
lagen implant in the prophylaxis and treatment of surgical
site infection in orthopaedic surgery. Int J Surg. 2012;10: of artesunate with paclitaxel loaded into poly-d,l-lactic-co-
S15–S20. glycolic acid nanoparticle for systemic delivery to exhibit
[19] Musters GD, Burger JWA, Buskens CJ, et al. Local applica- synergic chemotherapeutic response. Drug Dev Ind Pharm.
tion of gentamicin in the prophylaxis of perineal wound 2017;43:1952–1962.
infection after abdominoperineal resection: a systematic [37] Dargaville TR, Farrugia BL, Broadbent JA, et al. Sensors and
review. World J Surg. 2015;39:2786–2794. imaging for wound healing: A review. Biosens Bioelectron.
[20] Hussain ST. Local application of gentamicin-containing col- 2013;41:30–42.
lagen implant in the prophylaxis and treatment of surgical [38] Indian Pharmacopoeia. 6th Edition. Ghaziabad: Indian
site infection following vascular surgery. Int. J. Surg. 2012; Pharmacopoeia Commission; 2010.
10:S5–S9. [39] Bennison L, Miller C, Summers R, et al. The pH of wounds
[21] Nimesh S. Poly(D,L-lactide-co-glycolide)-based nanopar- during healing and infection: A descriptive literature review.
ticles. Gene Ther. 2013;309–329. Wound Pract Res. 2017; 25:63–69.
818 C. DHAL AND R. MISHRA

[40] Renuka M, Nishadh P, Jigar S, et al. Mucoadhesive wound [48] Naidu VGM, Madhusudhana K, Sashidhar RB, et al.
healing film of Doxycycline hydrochloride. Int J Drug Dev Polyelectrolyte complexes of gum kondagogu and chito-
Res. 2012;4:128–140. san, as diclofenac carriers. Carbohydr Polym. 2009;76:
[41] Himedia. Product Information [Internet]. [cited 2019 Feb 8]. 464–471.
Available from: http://himedialabs.com/TD/LA395.pdf [49] Bhowmick S, Koul V. Assessment of PVA/silver nanocompo-
[42] ICH Harmonised Tripartite Guidelines. Impurities: Guidelines site hydrogel patch as antimicrobial dressing scaffold:
for Residual Solvents. Q3C(R5) Current Step4. 2011. Synthesis, characterization and biological evaluation. Mater
[43] Cerqueira BBS, Lasham A, Shelling AN, et al. Development Sci Eng C. 2016;59:109–119.
of biodegradable PLGA nanoparticles surface engineered [50] Hines DJ, Kaplan DL. Poly(lactic-co-glycolic) acid-controlled-
with hyaluronic acid for targeted delivery of paclitaxel to
release systems: experimental and modeling insights. Crit
triple negative breast cancer cells. Mater Sci Eng C. 2017;
Rev Ther Drug Carrier Syst. 2013;30:257–276.
76:593–600.
[51] Zhang Y, Huo M, Zhou J, et al. DDSolver: An add-in pro-
[44] Tammam SN, Lamprecht A Nanostructures in drug delivery.
gram for modeling and comparison of drug dissolution
In: Cornier J, Owen A, Kwade A, et al., editors.
profiles. Aaps J. 2010;12:263–271.
Pharm. Nanotechnol. Innov. Prod. Wiley-VCH; 2017. Vol. 1.
[52] Bruschi ML. Strategies to modify the drug release from
p. 101–134.
[45] Iglesias M, Gonzalez-Olmos R, Manuel Goenaga J, et al. pharmaceutical systems: mathematical models of drug.
Phase behaviour of ethanol þ water þ ethyl acetate at Cambridge (UK): Woodhead Publishing Limited; 2015.
101.3 kPa. Phys Chem Liq. 2010;48:461–476. p. 63–86.
[46] Suhag A, Dixit J, Dhan P. Role of curcumin as a subgingival [53] Abbaszadegan A, Ghahramani Y, Gholami A, et al. The
irrigant: a pilot study. Periodontal Pract Today. 2007;4(2): effect of charge at the surface of silver nanoparticles on
115–121. antimicrobial activity against Gram-positive and Gram-nega-
[47] Rosenkrantz BE, Greco JR, Hoogerheide JG, et al. Gentamicin tive bacteria: A Preliminary Study. Exp Biol Med. 2014;2015:
sulfate. Anal Profiles Drug Subst. 1981;9:295–340. 1–8.