pharmaceutics

Article
A Smart Drug Delivery System Based on
Biodegradable Chitosan/Poly(allylamine
hydrochloride) Blend Films
Muhammad Sohail Sarwar 1,2,3 , Qingrong Huang 2 , Abdul Ghaffar 1, * , Muhmmad Amin Abid 4 ,
Muhammad Sohail Zafar 5 , Zohaib Khurshid 6 and Muhammad Latif 7, *
1 Department of Chemistry, University of Engineering and Technology, Lahore 54890, Pakistan;
sohailchemist77@gmail.com
2 Department of Food Science, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA;
qhuang@sebs.rutgers.edu
3 Department of Chemistry, Forman Christian College (A Chartered University), Lahore 54600, Pakistan
4 Department of Chemistry, University of Sahiwal, Sahiwal 57050, Pakistan; mabiduet@gmail.com
5 Department of Restorative Dentistry, College of Dentistry, Taibah University, Al Madinah,
Al Munawwarah 41311, Saudi Arabia; MZAFAR@taibahu.edu.sa
6 Department of Prosthodontics and Dental Implantology, College of Dentistry, King Faisal University,
Al-Ahsa 31982, Saudia Arabia; drzohaibkhurshid@gmail.com
7 Centre for Genetics and Inherited Diseases (CGID), Taibah University, Madinah Al Munawwarah 42318,
Saudi Arabia
* Correspondence: abdul.ghaffar@uet.edu.pk (A.G.); latifmayo@kaist.ac.kr (M.L.);
Tel.: +92-42-99029239-810 (A.G.); +966-14-8618888-77596 (M.L.)




Received: 11 December 2019; Accepted: 26 January 2020; Published: 4 February 2020


Abstract: The amalgamation of natural polysaccharides with synthetic polymers often produces
fruitful results in the area of drug delivery due to their biodegradable and biocompatible nature.
In this study, a series of blend films composed of chitosan (CS)/poly(allylamine hydrochloride)
(PAH) in different compositions were prepared as smart drug delivery matrices. The properties of
these polymeric films were then explored. Attenuated total reflectance-Fourier transform infrared
(ATR-FTIR) analysis confirmed an intermolecular hydrogen bonding between CS and PAH. Atomic
force microscopy (AFM) revealed improvements in surface morphology as the percentage of PAH in
the blend films increased up to 60% (w/w). Water contact angle (WCA) ranged between 97◦ to 115◦ ,
exhibiting the hydrophobic nature of the films. Two films were selected, CTH-1 (90% CS and 10%
PAH) and CTH-2 (80% CS and 20% PAH), to test for in vitro cumulative drug release (%) at 37 ± 0.5 ◦ C
as a function of time. It was revealed that for simulated gastric fluid (SGF) with pH 1.2, the cumulative
drug release (CDR) for CTH-1 and CTH-2 was around 88% and 85% in 50 min, respectively. Both
films converted into gel-like material after 30 min. On the other hand, in pH 7.4 phosphate buffer
saline (PBS) solution, the maximum CDR for CTH-1 and CTH-2 was 93% in 90 min and 98% in 120
min, respectively. After 120 min, these films became fragments. Sustained drug release was observed
in PBS, as compared to SGF, because of the poor stability of the films in the latter. These results
demonstrate the excellent potential of blend films in sustained-release drug delivery systems for
hydrophilic or unstable drugs.

Keywords: drug delivery platform; chitosan; natural and synthetic materials in drug delivery;
biodegradation; polymers; Ciprofloxacin; blend; film; anti-infective agents

Pharmaceutics 2020, 12, 131; doi:10.3390/pharmaceutics12020131 www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2020, 12, 131 2 of 18

1. Introduction
Natural biopolymers, especially polysaccharides, have attracted considerable interest in the
field of drug delivery due to their biodegradable, biocompatible, hydrophilic, and protective
properties [1–3]. In pharmaceutical research and applications, polymeric nano-particles and natural
biological macromolecules-based delivery systems are commonly used these days. These potential
carriers are capable of delivering bioactive compounds to specific cells and tissues with minimal
immune response at the nanoscale. Natural polymers, due to their diverse physico-chemical properties,
can be used to fabricate polymeric nano-carrier systems (NCS) that can optimize the bioavailability of
drugs by increasing retention time, minimizing side effects, increasing drug solubility, and reducing
dosage frequency [4].
Chitosan, a natural carbohydrate, is inexpensive, easily available, a cationic copolymer, and
can be obtained by deacetylation of chitin. It consists of N-acetyl-d-glucosamine and d-glucosamine
units. Considering the non-toxic, biocompatible, and biodegradable nature, chitosan is a prospective
candidate in the field of pharmaceutical industry [4,5]. Several researchers have investigated chitosan
in gene delivery, drug release, and biomedical areas [6–9]. It can increase the extent of drug delivery
across mucosal or nasal layers without causing any damage to the tissues [10]. On the contrary, various
processing conditions (such as a change in temperature, etc.) and environmental factors may trigger
degradation and deteriorate its polymeric structure [11].
It has been observed recently that two or more polymers are usually blended to obtain a wide range
of physico-chemical properties. Numerous properties of chitosan can be enhanced by blending with
natural polymers as well with synthetic ones such as zein, sodium alginate, curdlan, konjac glucomann,
poly(lactic acid), polycaprolactone, poly(ethylene oxide), poly(vinyl pyrollidine), and graphene
oxide [12–15]. Depending on the nature of biomedical applications, such as tissue engineering
and drug delivery, chitosan has been blended with polyethylene glycol fumarate, poly(ethylene
glycol) methyl ether, bone, polyethylene glycol, cartilage, skin, gelatin, dialdehyde starch, and
nerves [14,16–20].
Poly(allylamine hydrochloride) is a water-soluble weak-base, a cationic and biodegradable
polymer. It has a large number of amino groups that are present as a free amine or cationic ammonium
salt like other amine group-containing polymers [21–23]. Chitosan alone cannot satisfy biomedical
applications due to mechanical defects; therefore, blends of chitosan are employed to overcome
insufficient mechanical features [24–28]. Multitudinous systems comprising of polymeric materials
have been prepared to serve as drug carriers to investigate the controlled release behavior of payloads
and offer smart drug delivery. Polymeric biocompatible materials such as polyelectrolytes nanoparticles
coated with a bilayer of polyelectrolytes, namely PAH and poly (sodium 4-styerenesulfonate) (PSS)
were investigated for hydrophobic drug release [29]. It has been reported that PAH and polyurethane
multilayer films as a drug delivery system are potential contestants for pharmaceutical and biomedical
applications [22]. The efficiency of nanoparticulate drug delivery systems can be increased by modifying
poly(d,l-lactide-co-glycolide) via layer-by-layer adsorption, with two polyelectrolyte pairs such as
PAH/PSS and poly(l-lysine hydrobromide)/dextran sulfate [30].
Several efforts to develop functional materials (films, beads, and hydrogels) from chitosan have
been reported for potential use in wound healing [31] and drug delivery systems [32–37]. Blending
of natural polymers with synthetic polymeric material is an alternative approach to incorporate
a desired set of physico-mechanical characteristics in new materials. The film matrix generated
by the blending of two or more different polymeric systems generally results in modification
of physico-mechanical properties, compared to a film obtained by an individual polymer. For
instance, chitosan/starch blend films show higher flexibility and improved elongation [38], chitosan
and quaternized poly(4-vinyl-N-butyl) pyridine demonstrate robust tensile strength [39], blends of
chitosan/poly(vinylalcohol) [40], chitosan/N-methylol nylon 6 [41], and chitosan/polycaprolactone [12]
exhibit improved properties compared to pure chitosan films. Several publications related to the
Pharmaceutics 2020, 12, 131 3 of 18

blending of chitosan with natural (particularly cellulose and its derivative) [42–44] and synthetic
polymers [11,41,45–47] have been reported.
In continuation of our efforts to develop materials for controlled drug delivery [48,49], the purpose
of the current study was to develop a smart drug delivery system for ciprofloxacin hydrochloride
monohydrate (CPX) using biodegradable CS/PAH blend films. CS and PAH were selected to prepare
a series of blend films with different compositions as smart drug delivery matrices. The prepared
polymeric films were characterized to explore their properties. To study the drug release behaviour of
CS/PAH blend films, CPX was taken as a model drug. CPX is a fluoroquinolone drug that has a broad
antibiotic spectrum. It is widely used for microbial infections such as pulmonary, urinary, and dermal
infections [50] and anterior ocular infections [51]. It can be administered intravenously or orally [52]. It
is very effective against gram-positive and gram-negative bacteria that cause gastrointestinal, urinary
tract, abdominal, and respiratory infections. Prophylaxis and osteomyelitis, which is caused by P.
aeruginosa, has been treated with CPX [53]. Due to its low solubility, CPX is usually formulated as
a colloidal drug carrier, such as micelles, nano-suspensions, and polymeric nanoparticles [54]. CPX
offers bioavailability (60–80%) and serum half-life (~3–4 h); however, in clinical settings, repeated
administration (b.i.d, for 5 days) concerns have made CPX a suitable candidate for controlled-release
drug delivery system that can reduce gastric irritation and dose dumping concerns [55,56]. A number
of studies have shown the incorporation of CPX in polymeric matrices and have demonstrated control
release to function as wound-dressing materials [57–59], and antibacterial efficacy of CPX-loaded
imprinted hydrogels [60]. To further address the limitations of instantaneous release of CPX, the aim of
this study was to design and develop blend films using a biocompatible and biodegradable polymeric
blend of CS/PAH in varying compositions, to be used as a drug delivery matrix for sustained release.
We previously reported drug releasing films prepared by blending CS with methoxy polyethylene
glycol (mPEG) for controlled drug release applications [20]. In the present study, based on the
well-known fact that cationic polymers have the potential to adhere to negatively charged surfaces
of bacteria, we incorporated PAH primarily to impart flexibility in blend films and speculated on
the retention of the overall cationic nature of blend films. To the best of our literature survey and
knowledge, there are no publications reporting controlled release behaviour of CPX from CS/PAH
blended films as potential biomaterials for drug delivery applications.

2. Materials and Methods

2.1. Materials
Chitosan (Cat No. 448869, viscosity: 20–300 cP) with deacetylation degree (DDA) value ~75 to
85% molecular weight: 50 to 190 kDa was purchased from Sigma Aldrich, Saint Louis, MO, USA.
Poly(allylamine hydrochloride (PAH) (formula weight: 120,000~200,000 g·mol−1 was obtained from
Beantown Chemical, Hudson, NH, USA. Acetic acid (>99.9%) was also purchased from Sigma Aldrich,
Saint Louis, MO, USA. Ciprofloxacin hydrochloride monohydrate was collected from Tokyo Chemical
Industry Co. Ltd. (TCI, Portland, OR, USA). All of the other chemicals were used as received and
without any further purification.

2.2. Preparation of Films

Polymeric blend films of chitosan (CS) with Poly(allylamine hydrochloride) (PAH) were prepared
by solution casting technique as reported earlier [61]. The solutions in different CS/PAH ratios (100/0,
90/10, 80/20, 70/30, 60/40, 40/60) were prepared and each film was coded as CTH-0, CTH-1, CTH-2,
CTH-3, CTH-4, CTH-5, respectively. In the first step, chitosan was dissolved in acetic acid solution,
2% (v/v), with constant stirring for 90 min at room temperature. Secondly, an aqueous solution of
PAH, prepared in deionized water, was added in a dropwise manner into the chitosan solutions with
continuous stirring at room temperature. These blended solutions were stirred at room temperature for
90 min to obtain homogenous polymeric solutions. For film casting, the polymeric blended solutions
Pharmaceutics 2020, 12, 131 4 of 18

were poured into polystyrene petri dishes and allowed to dry under ambient conditions. The blend
films were peeled off and immersed in a solution of NaOH (1%), followed by thorough washing with
deionized distilled water. Finally, the prepared blended films were placed in an oven at 48 ± 2 ◦ C for 2
days, enclosed in airtight plastic bags, and stored at room temperature until further analysis.

2.3. Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy

For intermolecular determinations, ATR-FTIR spectra of the prepared films as well as PAH were
recorded using a spectrometer (Thermo Electron Corp., Madison, WI, USA). The spectra were taken in
transmittance mode ranging from 4000 to 400 cm−1 at a resolution of 4 cm−1 and 256 scans per sample.

2.4. Atomic Force Microscopy (AFM) Studies

Surface characterization of prepared films was subjected to AFM using Nanoscope IIIa Multimode
microscope (Veeco Instruments, Inc., Santa Barbara, CA, USA), equipped with silicon RTESP7 cantilever
(Veeco Nanoprobe, Camarillo, CA, USA) in tapping mode. For silicon tip, nominal spring constant
of 40 N/m was applied. AFM images were recorded as 10 µm × 10 µm using scan rate of 0.5 Hz in
air keeping ambient conditions. For AFM, blended solutions of polymers were spin-coated on newly
cleaved mica surface using a spin coater (Laurell Technologies Corporation, North Wales, PA, USA).
The spinning rate was kept at 1500 RPM for 2 min. All the prepared samples were placed in the oven
at 48 ± 2 ◦ C for two days to obtain complete dried films [13].

2.5. Water Contact Angle (WCA) Measurements

To calculate WCA in degree, the dried films were attached to the microscope glass slides using
double-sided tape. A water droplet (8 µL) was placed on each film by a micro-injector syringe
(Hamilton Company, Reno, NV, USA). Different images were recorded until WCA was constant using
video contact angle instrument VCA optima (AST Products, Inc., Billerica, MA, USA). An automatic
WCA measurement, from both the right and left-hand side of the drop, was done using VCA-optima
software. On average, twenty measurements were performed per film, which was cut from four
different sites.

2.6. Swelling Behaviour of Blend Films

The swelling experiments of pristine CS film and their blend films with PAH were conducted
in deionized water and ionic solutions, such as 0.1 M, 0.3 M, 0.5 M, 0.7 M, 0.9 M, 1.0 M NaCl, and
CaCl2 by following previously reported procedure [62]. All vacuum dried films were cut into smaller
pieces, weighed (~20 mg), and dipped into glass vials containing 40 mL of solvent/solution. Every 20
min, excess solvent was discarded, each vial was dried using tissue paper, and the weight of swollen
film was measured along with the vial. This procedure was repeated until equilibrium was achieved.
The experiment was repeated thrice for each film to calculate the standard deviation (SD). Degree of
swelling was calculated using Equation (1):

Ws − Wd
Swelling (g/g) = (1)
Wd

W d = weight of the dried films, W s = weight of the swollen films.

2.7. Cumulative Drug Release (CDR) Study

2.7.1. Preparation of Simulated Gastric Fluid (SGF) and Phosphate Buffer Saline (PBS) Solution
SGF (pH 1.2) was prepared by mixing NaCl (1 g) with 4 mL of HCl and diluted to 500 mL using
deionized water [62]. PBS (pH adjusted to 7.4 using 0.1 M NaOH and 0.1 M HCl) solution was prepared
by dissolving NaCl (8 g), KCl (0.2 g), Na2 HPO4 (1.44 g), and KH2 PO4 (0.24 g) in 800 mL deionized water
Pharmaceutics 2020, 12, 131 5 of 18

and diluted to 1000 mL [63]. A pH meter (Fischer Scientific accumet, Singapore 139949, Singapore)
was used to monitor the pH of solutions.

2.7.2. Drug Loading Method

To study controlled drug release, 20 mg of CPX was dissolved in deionized water. The drug
solution was added dropwise in a polymeric blended solution of CS/PAH (90/10). The solution was
stirred for 90 min at room temperature prior to pouring into a polystyrene petri dish for film formation
under ambient conditions. CPX was also loaded in a film with CS/PAH (80/20) by repeating the same
procedure as mentioned above. Finally, the prepared blended films were peeled off, enclosed in airtight
plastic bags, and stored at room temperature in the dark until further analysis.

2.7.3. In Vitro Drug Release

The CDR mechanism was studied using a smart dissolution tester (Sortax AG, CH-4123, Allschwil 1,
CE 7smart, Basel, Switzerland) [64]. The average thickness of drug-loaded films was 40–70 µm,
determined using a micrometre (Mitutoyo Corporation, model PK-0505CPX, Kanagawa, Japan) by
performing three measurements for each specimen. Films were cut from six different regions for
triplicate measurements in SGF and PBS solutions. The average weight of the drug-loaded CTH-1
and CTH-2 films was 0.0791 g and 0.0588 g, respectively. These drug-loaded films were put into a
500-mL dissolution medium, such as SGF and PBS solution, at temperature 37 ± 0.5 ◦ C and stirred at
50 rpm. Every 10 min, 5 mL solution was collected separately from each SGF and PBS solution using a
pipette. The obtained solutions were filtered through a membrane (pore size; 0.45 µm). Fresh 5 mL
of the SGF and PBS solutions were added back every time to keep the volume at 500 mL. The drug
release analysis was investigated for 2–3 h in the SGF and PBS solutions. UV scans of the collected
solutions were recorded at λmax 277 nm for SGF and 270 nm for PBS using a UV–Visible spectrometer
(Agilent Technologies, Cary 60, UV–Vis, Santa Clara, CA, USA). The standard drug solutions of CPX
(100 ppm) in SGF and PBS were used as a reference [62].

2.8. Statistical Analysis

Data were handled using the SPSS (v.20, IBM, USA) and expressed in the form of means ± standard
deviation (SD). The data were analyzed statistically by the two-way analysis of variance (ANOVA) and
p < 0.05 was considered as statistically significant. Independent sample t-test was applied to compare
drug release in different two media, SGF and PBS.

3. Results and Discussion

3.1. Preliminary Characterization

We prepared polymeric blend films of CS/PAH using the solution casting technique. A solution of
chitosan in acetic acid (2% v/v) and an aqueous solution of PAH were prepared separately. The aqueous
solution was then added drop by drop to the chitosan solution. A preliminary examination showed
that the prepared films were translucent and slightly yellowish in color. Films thickness (~40–70 µm)
was determined using a micrometre (Mitutoyo Corporation, model PK-0505CPX, Kanagawa, Japan) by
performing three measurements per specimen (Table 1). We could not find any statistically significant
difference in film thickness among the six samples (CTH0-CTH5) using ANOVA, F(5,18) = 2.35, p-value
= 0.083. The projected interactions between CS and PAH are shown in Figure 1.
Pharmaceutics 2020, 12, 131 6 of 18

Pharmaceutics 2020, 12, x 6 of 18

Table 1. Films thickness data of each film from different regions.
Table 1. Films thickness data of each film from different regions.
Films Films Films Films Average
Films
Thickness Films
Thickness Films
Thickness Films
Thickness Average
Films
Films * SD (±)
(1st region) Thickness
Thickness (4th region)
(3rd region) Thickness
(2nd region) Thickness Thickness
Films
Films (µm) (µm) (µm) (µm) (µm) * SD (±)
(1st region) (2nd region) (3rd region) (4th region) Thickness
CTH-0 (µm)40 (µm)50 (µm) 70 (µm) 50 (µm)
52.5 10.9
CTH-0
CTH-1 4070 5080 7090 5040 52.5
70 10.9
18.7
CTH-2
CTH-1 7040 8040 9050 40 60 47.5
70 8.3
18.7
CTH-3
CTH-2 4050 4070 5040 6050 52.5
47.5 10.9
8.3
CTH-4
CTH-3 5070 7070 4040 5070 62.5
52.5 13.0
10.9
CTH-5
CTH-4 7050 7040 40 30 70 40 40
62.5 7.1
13.0
CTH-5 * SD means
50 standard deviation,
40 CTH stands 30
for chitosan/poly(allylamine
40 hydrochloride)
40 blend. 7.1
* SD means standard deviation, CTH stands for chitosan/poly(allylamine hydrochloride) blend.

Figure 1.
Figure The proposed
1. The proposed interactions
interactions between
between the
the CS
CS and
and PAH
PAH in
in the
the prepared
prepared blend
blend films.
films.
3.2. ATR-FTIR Spectroscopy
3.2. ATR-FTIR Spectroscopy
FTIR spectroscopy is a technique frequently employed to uncover intermolecular interactions,
FTIR spectroscopy
particularly is a technique
hydrogen bonding between frequently employed
diverse types to uncover
of functional intermolecular
groups interactions,
in polymeric molecules.
Intermolecular interactions either lead to a shift in frequency or band broadening of particularmolecules.
particularly hydrogen bonding between diverse types of functional groups in polymeric functional
Intermolecular interactions either lead to a shift in frequency or band broadening
groups. Information related to the conformational changes occurring in compatible blends of particular
can be
acquired by FTIR spectral analysis [65]. As we had prepared several solutions—CTH-0 (100/0), blends
functional groups. Information related to the conformational changes occurring in compatible CTH-1
can be acquired
(90/10), by FTIRCTH-3
CTH-2 (80/20), spectral(70/30),
analysis [65]. As
CTH-4 we had
(60/40), andprepared
CTH-5several solutions—CTH-0
(40/60)—of (100/0),
CS/PAH in different
CTH-1 we
ratios, (90/10), CTH-2 (80/20),
investigated whetherCTH-3 (70/30), CTH-4
CS molecules (60/40), and
really interacted CTH-5
with (40/60)—ofbackbone
the polymeric CS/PAH of in
different ratios, we investigated whether CS molecules really interacted with the polymeric backbone
of PAH. To confirm possible interactions, the prepared biodegradable blend films—chitosan and
PAH samples—were subjected to ATR-FTIR spectroscopy. The presence of each component was
PAH, and their blend films are shown in Figure 2. In the case of PAH spectra, the bands at 1610 cm−1
and 1381 cm−1 were detected corresponding to N–H in the plane bending motion and C–H bending,
respectively [66]. The broadband varying from 3400 to 2800 cm−1 with two split peaks was observed—
one peak at 2910 cm−1 and 2845 cm−1, attributed to C–H asymmetric and symmetric vibrations—while
Pharmaceutics 2020, 12, 131 7 of 18
another sharp peak at 3391 cm−1 was caused by stretching vibration of the intermolecular as well as
intramolecular N–H bond of the primary amine group. These peaks were observed in all blend films,
but with
PAH. less intensity
To confirm [21].interactions, the prepared biodegradable blend films—chitosan and PAH
possible
In the spectra of chitosan,
samples—were subjected to ATR-FTIR bands of amide-I (C=O)
spectroscopy. Theand amide-II
presence (N–H)
of each were detected
component at around
was established
1640
by thecm
−1 and 1538ofcm
appearance
−1 , respectively.
absorption Thedifferent
bands for cis-amide III bandgroups.
functional was located at 1248ofcm
The spectra CS,[62,67].
−1 A peak
PAH, and their
at 1078 cm −1 confirmed C–O–C stretching vibrations [68]. The glycosidic linkages appeared
blend films are shown in Figure 2. In the case of PAH spectra, the bands at 1610 cm and 1381 −1 at cm
1154
−1
and 894
were cm . Moreover,
detected
−1
corresponding a band ranging
to N–H from
in the 3391
plane to 3386motion
bending cm was
−1
andassigned to therespectively
C–H bending, O–H stretching
[66].
of chitosan [6,69]. We noticed that as the concentration
−1 of PAH in blend films
The broadband varying from 3400 to 2800 cm with two split peaks was observed—one peak at 2910 increased, the intensity
of the
cm band
−1 and alsocm
2845 decreased. In the
−1 , attributed typical
to C–H spectrum and
asymmetric of a symmetric
CS/PAH composite film, the amino
vibrations—while anotherpeak
sharpof
CS shifts
peak at 3391fromcm1541
−1 was tocaused
1510 cm −1 and the intensity
by stretching vibrationofofthese bands decrease
the intermolecular dramatically
as well with the
as intramolecular
addition of PAH, indicating an intermolecular hydrogen bonding between
N–H bond of the primary amine group. These peaks were observed in all blend films, but with lessCS and PAH (Figure 2)
[16]. This manifests
intensity [21]. that CS has good compatibility with PAH.

Figure 2. ATR-FTIR spectra of chitosan (CTH-0), PAH (CTH-6), and their blend films showing different
Figure 2. ATR-FTIR spectra of chitosan (CTH-0), PAH (CTH-6), and their blend films showing different
chitosan/PAH compositions—100/0 (CTH-0), 80/10 (CTH-2), 60/40 (CTH-4), and 40/60 (CTH-5).
chitosan/PAH compositions—100/0 (CTH-0), 80/10 (CTH-2), 60/40 (CTH-4), and 40/60 (CTH-5).
In the spectra of chitosan, bands of amide-I (C=O) and amide-II (N–H) were detected at around
3.3. Atomic Force Microscopy Study
1640 cm−1 and 1538 cm−1 , respectively. The cis-amide III band was located at 1248 cm−1 [62,67]. A peak
at 1078
AFM cm−1is confirmed C–O–C
an excellent stretching
tool that vibrations
investigates the [68]. Theand
texture glycosidic linkages
morphology of appeared at 1154
diverse surfaces,
and −1 . Moreover, a band ranging from 3391 to 3386 cm−1 was assigned to the O–H stretching
894 cmthin
including films [70–72]. The versatility of AFM allows acquiring of rigorous observations and
of chitosan [6,69].
assessment We noticed
of the textural and that as the concentration
morphological of PAH
characteristics in blend
of films. films
In this increased,
study, the intensity
the surface texture
of
andtheroughness
band alsoof decreased.
CS and itsInblend
the typical
films spectrum
with PAHof(CTH-0,
a CS/PAH composite
CTH-1, CTH-2, film, the amino
CTH-3, CTH-4,peak of CS
CTH-5)
shifts from 1541 using
were examined cm−1with
to 1510AFM and athe intensity of
Nanoscope these
IIIa bands decrease
Multimode dramatically
microscope with the addition
(Veeco Instruments, Inc.,
of PAH,
Santa indicating
Barbara, CA, an intermolecular
USA), equipped with hydrogen
a siliconbonding
RTESP7between CS(Veeco
cantilever and PAH (Figure 2)Camarillo,
Nanoprobe, [16]. This
manifests
CA, USA) that CS hasmode.
in tapping good compatibility
For the siliconwith PAH.
tip, nominal spring constant of 40 N/m was applied. AFM
images were recorded as 10 μm × 10 μm using scan rate of 0.5 Hz under atmospheric conditions at
3.3. Atomic
ambient Force Microscopy
temperature. For AFMStudy
measurements, the blended solutions of polymers were spin-coated
on a AFM
newly cleaved mica surface using a spin
is an excellent tool that investigates thecoater
texture(Laurell Technologies
and morphology Corporation,
of diverse surfaces,USA) and
including
the spinning
thin rate was
films [70–72]. Themaintained
versatility ofatAFM
1500 allows
RPM for 2 min. Experimental
acquiring data were processed
of rigorous observations with the
and assessment of
the textural and morphological characteristics of films. In this study, the surface texture and roughness
of CS and its blend films with PAH (CTH-0, CTH-1, CTH-2, CTH-3, CTH-4, CTH-5) were examined
using AFM with a Nanoscope IIIa Multimode microscope (Veeco Instruments, Inc., Santa Barbara, CA,
USA), equipped with a silicon RTESP7 cantilever (Veeco Nanoprobe, Camarillo, CA, USA) in tapping
mode. For the silicon tip, nominal spring constant of 40 N/m was applied. AFM images were recorded
Pharmaceutics 2020, 12, 131 8 of 18

as 10 µm × 10 µm using scan rate of 0.5 Hz under atmospheric conditions at ambient temperature.

For AFM measurements,
Pharmaceutics 2020, 12, x the blended solutions of polymers were spin-coated on a newly cleaved 8 of 18
mica surface using a spin coater (Laurell Technologies Corporation, USA) and the spinning rate was
help of Nanoscope
maintained at 1500 5.30
RPMsoftware [13].
for 2 min. The micrographs
Experimental infer that
data were films coded
processed with thewith CTH-0
help and CTH-
of Nanoscope
2 were
5.30 found[13].
software to beTherough. On the other
micrographs inferhand, CTH-4
that films andwith
coded CTH-5 filmsand
CTH-0 displayed a smooth
CTH-2 were foundsurface
to be
morphology (Figure 3). This might be attributed to the rigid and crystalline
rough. On the other hand, CTH-4 and CTH-5 films displayed a smooth surface morphology (Figurenature of chitosan [73].
3).
These properties of chitosan molecules were also affected by sample preparation.
This might be attributed to the rigid and crystalline nature of chitosan [73]. These properties of Like these films,
CTH-0 and
chitosan CTH-2 present
molecules were alsospherical,
affectedbranch crystals
by sample due to theLike
preparation. higher percentage
these of chitosan
films, CTH-0 100%
and CTH-2
(w/w) and 80% (w/w), respectively [69]. AFM analysis showed that as the concentration
present spherical, branch crystals due to the higher percentage of chitosan 100% (w/w) and 80% (w/w), of PAH
increased up
respectively to AFM
[69]. 60% analysis
(w/w), the surface
showed that morphology improved,
as the concentration of PAH representing
increased up antoincrease
60% (w/w),in
compatibility with polymer chains. The CTH-5 film was found to be more homogenous
the surface morphology improved, representing an increase in compatibility with polymer chains. The as compared
to the other
CTH-5 films.
film was found to be more homogenous as compared to the other films.

Figure 3. AFM images of films with different chitosan/PAH compositions—100/0 (CTH-0), 80/10
Figure 3.60/40
(CTH-2), AFM(CTH-4),
images of films
and 40/60with different chitosan/PAH
(CTH-5)—and compositions—100/0
their corresponding (CTH-0),
3D images in panels on 80/10
mica
(CTH-2), 60/40 (CTH-4), and 40/60 (CTH-5)—and their corresponding 3D images in panels on mica
using tapping mode. The scale size was 10 × 10 µm and the z scale was 20 nm and 30 nm, respectively.
using tapping mode. The scale size was 10 × 10 μm and the z scale was 20 nm and 30 nm, respectively.
3.4. WCA Measurements
3.4. WCA Measurements
Water contact angle measurement is one of the most common methodologies used to determine
Water contact
the wettability of a angle measurement
surface/material andistheir
one of the most common
hydrophobic methodologies
or hydrophilic used The
nature [74]. to determine
intrinsic
the wettability
values of WCA of area dependent
surface/material and their
on surface hydrophobic
roughness, porosity,or hydrophilic nature [75].
and heterogeneity [74]. We
Therecorded
intrinsic
valuesofofdifferent
WCA WCA are dependent
images for eachonblend
surfacefilmroughness,
using video porosity,
contactandangleheterogeneity
instrument VCA [75]. We recorded
optima (AST
WCA of different
Products, imagesMA,
Inc. Billerica, for each
USA)blend
until film
WCA using video
became contact angle
constant. instrument
An automatic WCA VCA optima (AST
measurement,
Products,
from both Inc. Billerica,
the right and MA, USA)side
left-hand untilof WCA
the drop,became
was constant. An automaticsoftware.
done by VCA-optima WCA measurement,
On average,
from both
twenty the right andwere
measurements left-hand side ofper
performed thefilm,
drop,which
was done
was cutby VCA-optima software.
from four different OnA
sites. average,
report
twenty measurements
demonstrated werecontact
that the water performed
angleper film, which
of blank chitosan was
wascut fromthan
higher ◦ due to its
four83different sites. A report
hydrophilic
demonstrated
nature and highthat thetension
surface water [76].
contact angleto of
In order blank the
decrease chitosan was higher
hydrophilicity than 83°
of chitosan, due
it was to its
blended
hydrophilic
with PAH tonature
impartand high surface
flexibility. tension
The WCA of[76]. In order tofilms
the prepared decrease the hydrophilicity
fluctuated between 96.7of to ◦ 114.7◦
chitosan,
it was blended
because with PAHratio
of a CS-to-PAH to impart flexibility.
that varied as shownThe WCA of the
in Figure 4. prepared
As shownfilms fluctuated
in Table 2, the WCAbetweenof
96.7° toCTH-1,
CTH-0, 114.7° because of a CS-to-PAH
CTH-2, CTH-3, CTH-4, andratio that varied
CTH-5 was 107as±shown ◦ in Figure
3.5 , 114.7 ◦
± 2.0 ,4.112
As±shown ◦ in Table
3.9 , 108.6 ◦,
± 1.82,
the WCA
96.7 ◦
± 2.5 of CTH-0,
, and 110.8CTH-1, ◦
± 2.5 , CTH-2, CTH-3,
respectively. CTH-4,
It was notedand
thatCTH-5
as thewas 107 ± 3.5°, 114.7
concentration of PAH ± 2.0°, 112 ±films
in blend 3.9°,
108.6 ± 1.8°,
increased up96.7 ± 2.5°,
to 60% and except
(w/w), 110.8 ±40%
2.5°,(w/w),
respectively.
the WCA It was noted that
increased. Thisasincrease
the concentration
might be due of PAH
to a
in blend films
decrease increased
in intensity up togroups,
of polar 60% (w/w), except
resulting in 40%
more(w/w), the WCA
networking increased.
among polymer This increase
chains [9]. might
be due to a decrease in intensity of polar groups, resulting in more networking among polymer chains
[9].
 CTH-0 107.4 3.5
CTH-1 114.7 2.0
CTH-2 112.0 3.9
CTH-3 108.6 1.8
CTH-4 96.7 2.5
Pharmaceutics 2020,CTH-5
12, 131 110.8 2.5 9 of 18
* SD means standard deviation; all experiments were performed in triplicate.

Figure 4. Water contact angles of films with chitosan/PAH ratios of 100/0 (CTH-0), 90/10 (CTH-1), 80/10
Figure 4. Water contact angles of films with chitosan/PAH ratios of 100/0 (CTH-0), 90/10 (CTH-1),
(CTH-2), 70/30 (CTH-3), 60/40 (CTH-4), and 40/60 (CTH-5).
80/10 (CTH-2), 70/30 (CTH-3), 60/40 (CTH-4), and 40/60 (CTH-5).
Table 2. Water contact angles data of films having CS/PAH ratios of 100/0 (CTH-0), 90/10 (CTH-1), 80/10
3.5. Swelling
(CTH-2),Behaviour in Different
70/30 (CTH-3), Media and 40/60 (CTH-5).
60/40 (CTH-4),

Films
3.5.1. Swelling in Deionized Water Contact Angle (degree)
Water * SD (±)
CTH-0 107.4 3.5
The swelling CTH-1
behaviour of pristine CS and CSPAH blend films was studied
114.7 2.0
as a function of time
(Table 3). It was observed
CTH-2 that all films displayed
112.0 a linear increase with time 3.9and equilibrium for the
CTH-1, CTH-2, and CTH-4 films were attained
CTH-3 108.6at around 80 min, 60 min, and 1.8 80 min, respectively.
The CTH-3 and CTH-5CTH-4films exhibited a distinctive
96.7 behaviour as these films 2.5 were converted into a
CTH-5
gel-like material after 25 min and 40 min, 110.8 respectively. The CTH-1 film2.5 had a higher degree of
* SD means standard deviation; all experiments were performed in triplicate.
swelling response—around 122 g/g after 80 min—as compared to the other blend films, but less than
blank chitosan film, which was around 182 g/g (Figure 5).
3.5. Swelling Behaviour in Different Media
Table 3. Swelling data of CS/PAH films in deionized water.
3.5.1. Swelling in Deionized Water
CTH-0 CTH-1 CTH-2 CTH-3 CTH-4 CTH-5
Time
(min)
The swelling
Swelling behaviour
SD of
Swelling pristine
SD CS and
Swelling CSPAH
SD blend
Swellingfilms
SDwas studied
Swelling as
SDa function
Swellingof time
SD
(g/g)
(Table 3). It (±)
was observed (g/g) (±)
that all films (g/g)
displayed (±)
a linear(g/g)
increase(±)with time
(g/g) and (±) (g/g) for(±)
equilibrium the
20 109.96 7.06 99.15 3.49 55.90 6.42 90.00 6.70 24.20 3.43 41.90 4.56
CTH-1, CTH-2, and CTH-4 films were attained at around 80 min, 60 min, and 80 min, respectively.
40 136.41 6.25 106.49 3.71 97.26 3.59 92.00 9.70 25.30 3.77 46.12 9.59
The60 CTH-3 and CTH-5
158.62 5.95 films
119.52exhibited
3.67 a97.27
distinctive
7.51 behaviour
-- as--these 45.32
films were
1.04 converted
-- into
-- a
gel-like
80 material
181.39 after
6.70 25 min
122.40and 40
0.57 min, respectively.
95.86 6.52 The CTH-1
-- film
-- had a higher
47.42 degree--of swelling
3.21 --
100 181.95 7.08 106.29 2.27 98.14 4.18 -- -- 47.35
response—around 122 g/g after 80 min—as compared to the other blend films, but less than blank 2.33 -- --
120 -- -- -- -- 98.44 3.19 -- -- 46.14 1.09 -- --
chitosan film, which was around 182 g/g (Figure 5).
140 -- -- -- -- 98.99 6.22 -- -- 45.59 2.46 -- --
SD means standard deviation; in order to obtain standard deviation, the experiment was repeated
Table 3. Swelling data of CS/PAH films in deionized water.
three times for each film.
Time CTH-0 CTH-1 CTH-2 CTH-3 CTH-4 CTH-5
(min)
Swelling SD Swelling SD Swelling SD Swelling SD Swelling SD Swelling SD
(g/g) (±) (g/g) (±) (g/g) (±) (g/g) (±) (g/g) (±) (g/g) (±)
20 109.96 7.06 99.15 3.49 55.90 6.42 90.00 6.70 24.20 3.43 41.90 4.56
40 136.41 6.25 106.49 3.71 97.26 3.59 92.00 9.70 25.30 3.77 46.12 9.59
60 158.62 5.95 119.52 3.67 97.27 7.51 – – 45.32 1.04 – –
80 181.39 6.70 122.40 0.57 95.86 6.52 – – 47.42 3.21 – –
100 181.95 7.08 106.29 2.27 98.14 4.18 – – 47.35 2.33 – –
120 – – – – 98.44 3.19 – – 46.14 1.09 – –
140 – – – – 98.99 6.22 – – 45.59 2.46 – –
SD means standard deviation; in order to obtain standard deviation, the experiment was repeated three times for
each film.
 Pharmaceutics 2020, 12, 131 10 of 18
Pharmaceutics 2020, 12, x 10 of 18
Pharmaceutics 2020, 12, x 10 of 18

Figure 5. Swellingbehaviour
Figure behaviour of CS/PAHfilms
films withdifferent
different compositionsin
in deionizedwater.
water.
Figure5.5.Swelling
Swelling behaviourofofCS/PAH
CS/PAH filmswith
with differentcompositions
compositions indeionized
deionized water.
Theswelling
The swelling of blendfilms
films wasdue
due to diffusionof
of waterfrom
from theextracellular
extracellular matrixtotothe
the
The swellingofofblend
blend filmswas
was duetotodiffusion
diffusion ofwater
water fromthethe extracellularmatrix
matrix to the
polymeric
polymeric structures [77]. This mode of swelling was calculated by the following equation:
polymericstructures
structures[77].
[77].This
Thismode
modeofofswelling
swellingwas
wascalculated
calculatedby
bythe
thefollowing
followingequation:
equation:
FFF===ktnn (2)
kt
ktn (2)
(2)
where,
where,
where,
kk==swelling rate constant describing the characteristics ofofpolymer network and water
k = swelling
swelling rate
rate constant
constant describing
describing the the characteristics
characteristics of polymer
polymer network
network and and water
water
nn==swelling
swelling or
or diffusional
diffusional exponent
exponent
n = swelling or diffusional exponent
FF==fractional swelling atatthe time (t), calculated by the swelling ratio
ratioof
ofWWt tt(swelling ratio atattime
F = fractional
fractional swelling
swelling at the the time
time (t),
(t), calculated
calculated by by the
the swelling
swelling ratio of W (swelling
(swelling ratio
ratio at time
time
t)t)and
and WW eq (swelling ratio at equilibrium time t).
Weq (swelling ratio at equilibrium time t).
t) and eq (swelling ratio at equilibrium time t).
In
Inorder
orderto toobtain the
obtainthe values
thevalues
valuesof ofofnnand
and k,k,the
theswelling
swelling data ofofpure CS
CSand its
itsblend films was
In order to obtain n and k, the swelling datadata
of purepure
CS and and
its blend blend
films films was
was used.
used.
used. Figure
Figure 66showed
showed aa plot
plot between
between ln(F)
ln(F) versus
versus ln(t)
ln(t)and
and the
the values
valuesofofdiffusion
diffusion parameters
parameters (Table
(Table
Figure 6 showed a plot between ln(F) versus ln(t) and the values of diffusion parameters (Table 4).
4).
4).It
Itcan
can be inferred from Table 44that nn<<0.5 (corresponding totoFickian
Fickiantransport)
transport)for forall
allfilms,
films,which
It can be be inferred
inferred from
from Table
Table 4 thatthatn< 0.50.5(corresponding
(correspondingto Fickian transport) for all films, which
which
indicated
indicated that
that the
therate
rate of
of relaxation
relaxation was
was higher
higher than
thanthe
the rate
rateof
of diffusion.
diffusion.
indicated that the rate of relaxation was higher than the rate of diffusion.

Figure 6. Swellingbehavior
Figure behavior of CS/PAHfilms
films withdifferent
different compositions.
Figure6.6.Swelling
Swelling behaviorofofCS/PAH
CS/PAH filmswith
with differentcompositions.
compositions.

Table
Table4.4.Diffusion
Diffusionparameters
parametersofofdifferent
differentCS/PAH
CS/PAHfilms.
films.
Parameters
Parameters CTH-0
CTH-0 CTH-1
CTH-1 CTH-2
CTH-2 CTH-3
CTH-3 CTH-4
CTH-4 CTH-5
CTH-5
nn 0.355
0.355 0.160
0.160 0.280
0.280 0.098
0.098 0.442
0.442 0.138
0.138
Intercept
Intercept −−1.58
1.58 −−0.70
0.70 −−1.25
1.25 −−0.31
0.31 −−2.040
2.040 −−0.51
0.51
kk 0.457
0.457 −−0.357
0.357
0.223
0.223 −−1.204
1.204
0.713
0.713 −−0.673
0.673
The
Theexperiment
experimentwas
wasrepeated
repeatedthree
threetimes
timesfor
foreach
eachfilm.
film.
Pharmaceutics 2020, 12, x 11 of 18

3.5.2. Swelling Behaviour in Ionic Solutions

The swelling behaviour of pure CS and CS/PAH blend films as a function of time was evaluated
Pharmaceutics 2020, 12, 131 11 of 18
in different ionic solutions, such as NaCl and CaCl2. The effect of the ionic concentration of
electrolytes on the swelling behavior of blend films is shown in Figures 7 and 8. These Figures
demonstrate that with the Table 4. Diffusion
increase parameters of
of concentration ofdifferent
electrolyteCS/PAH films. medium, the swelling
in swelling
capacity of blend films significantly decreased. It was found that the nature of salt and the
Parameters CTH-0 CTH-1 CTH-2 CTH-3 CTH-4 CTH-5
concentration of ions had a noticeable influence on the swelling response. As the concentration of
n 0.355
both electrolytes (NaCl and CaCl0.160 0.280 0.098 0.442
2) increased, the rate of swelling decreased (Tables 5 and 6). This
0.138
Intercept −1.58 −0.70 −1.25 −0.31 −2.040 −0.51
might be k due to the0.457 generation of−0.357high-charge screening0.223 effects, which ultimately
−1.204 0.713 reduced −0.673
the rate of
diffusion and osmotic pressure developed between the films and the external solvent. Consequently,
The experiment was repeated three times for each film.
the diffusion of solvent into blend film decreased, resulting in lesser swelling. From Figures 7 and 8,
it can be inferred that maximum swelling was shown by CTH-1 in NaCl (0.1 M) compared to CaCl2
3.5.2. Swelling Behaviour in Ionic Solutions
(0.1 M). The swelling value for CTH-1 was 41 g/g and 26 g/g in NaCl (0.1 M) and CaCl2 (0.1 M),
respectively [77]. behaviour
The swelling Furthermore, overall
of pure higher
CS and swelling
CS/PAH blendresponse
films aswas noticedofintime
a function NaClwassolution,
evaluated as
compared
in different to other
ionic CaCl2 solutions.
solutions, such as NaCl It wasandobserved
CaCl2 . The thateffect
the CTH-3 film concentration
of the ionic was convertedofinto gel-like
electrolytes
material
on in 0.1 Mbehavior
the swelling and 0.3 of M blend
CaCl2 solutions.
films is shown in Figures 7 and 8. These Figures demonstrate that
with the increase of concentration of electrolyte in swelling medium, the swelling capacity of blend
filmsTable 5. Data showing
significantly swelling
decreased. behaviour
It was found of CS/PAH
that the naturefilms ofin different
salt and molar concentrationsofofions
the concentration NaCl.
had a
noticeable influence
CTH-0 on the swelling
CTH-1 response.CTH-2As the concentration
CTH-3 of both electrolytes
CTH-4 (NaCl
CTH-5 and
CaCl2 ) increased,
[NaCl] Swelling the SDrateSwelling
of swelling SD decreased
Swelling (Tables
SD 5Swelling
and 6). This
SD might Swellingbe dueSDto the generation
Swelling SD
(g/g) (±) (g/g) (±) (g/g) (±) (g/g)
of high-charge screening effects, which ultimately reduced the rate(±)of diffusion
(g/g)
and(±) (g/g)
osmotic (±)
pressure
0.1 M 27.25 1.95 40.69 1.09 21.32 0.00 33.84 1.62 14.53 1.96 22.33 0.23
developed
0.3 M between
23.58 the films
1.14 and the
31.04 external
2.28 15.98solvent.
1.53 Consequently,
20.56 0.07the diffusion
10.33 of
1.69solvent into blend
14.54 1.48
film decreased,
0.5 M 14.95 resulting
0.95 in lesser 1.51
24.66 swelling. 12.86From0.07Figures18.65 7 and 8, it can 9.74
1.04 be inferred
1.52 that maximum
10.70 0.38
0.7 M 14.56 0.76 16.75
swelling was shown by CTH-1 in 0.52
NaCl (0.1 10.59 1.95
M) compared 11.70
to CaCl 0.93 8.78 1.24 10.03
2 (0.1 M). The swelling value for
0.50
0.9 M 2.11 0.92 10.38 1.22 9.34 1.40 10.56 1.49 8.12 1.73 10.07 1.24
CTH-1
1.0 M was 0.84
41 g/g and0.8326 g/g in NaCl
1.79 (0.1 M)
1.02 and CaCl
8.10 0.59 2 (0.17.85
M), respectively
1.74 [77]. Furthermore,
8.14 0.17 5.27 overall
1.07
higher SDswelling response
means standard was noticed
deviation; in order in NaCl solution,
to obtain standard as deviation,
comparedthe to experiment
other CaClwas 2 solutions.
performed It was
observed that the CTH-3 film
in triplicate for each film. was converted into gel-like material in 0.1 M and 0.3 M CaCl 2 solutions.

Figure 7.
Figure Swelling behavior
7. Swelling behavior of
of CS/PAH
CS/PAH films
films in
in NaCl
NaCl solutions
solutions of
of variable
variable molar
molar concentrations.
concentrations.
 0.1 M 30.95 0.99 25.54 0.99 18.69 0.56 - - 24.83 0.84 23.87 1.21
0.3 M 14.55 0.49 17.22 1.26 12.16 1.63 - - 8.66 1.06 13.96 0.74
0.5 M 7.04 0.90 9.50 1.59 10.82 0.69 8.91 1.69 7.48 0.15 7.51 0.56
0.7 M 8.33 1.50 10.38 1.59 9.06 0.57 8.10 0.01 6.76 0.37 10.40 1.63
0.9 M 5.67 0.98 10.06 0.97 9.18 1.89 7.09 0.75 8.82 0.18 11.70 0.62
1.0 M 7.11 0.25 11.88 1.07 7.80 0.68 4.42 0.26 11.17 2.86 8.15 1.23
Pharmaceutics 2020, 12, 131 12 of 18
SD means standard deviation; in order to obtain standard deviation, the experiment was repeated
three times for each film.

Figure 8.
Figure Swellingbehaviour
8. Swelling behaviourof
ofCS/PAH
CS/PAH films in different molar concentrations of CaCl22 solutions.

Table 5. Data showing swelling behaviour of CS/PAH films in different molar concentrations of NaCl.
3.6. Cumulative Drug Release (CDR) Study
CTH-0 CTH-1 CTH-2 CTH-3 CTH-4 CTH-5
[NaCl]
Ciprofloxacin hydrochloride monohydrate, a model drug, was loaded in two films coded as
Swelling SD Swelling SD Swelling SD Swelling SD Swelling SD Swelling SD
CTH-1 and CTH-2
(g/g) (Table
(±) 7).(g/g)
Based on
(±) encouraging
(g/g) results(g/g)
(±) as described
(±) in Sections
(g/g) (±) 3.1–3.5,
(g/g) the CTH-
(±)
1 and
0.1 MCTH-227.25 blend films
1.95 were40.69selected.
1.09 These
21.32 two 0.00
blend films
33.84 had 1.62a greater
14.53 concentration
1.96 22.33of natural
0.23
0.3 M 23.58 1.14 31.04 2.28 15.98 1.53 20.56 0.07 10.33 1.69 14.54 1.48
polymer
0.5 M
used in
14.95
the present
0.95
study
24.66
(chitosan).
1.51 12.86
However,
0.07
CTH-3
18.65
and
1.04
CTH-59.74
showed
1.52
more
10.70
solubility
0.38
in aqueous
0.7 M media
14.56 and
0.76CTH-416.75film had
0.52 a greater
10.59 concentration
1.95 11.70 of0.93
PAH. The 8.78 controlled
1.24 drug release
10.03 0.50
0.9 M 2.11 0.92 10.38 1.22 9.34 1.40 10.56 1.49 8.12 1.73 10.07 1.24
from1.0 M
both films0.84
(CTH-1
0.83
and1.79
CTH-2)1.02was studied
8.10
in SGF (pH
0.59 7.85
1.2)1.74
as well8.14as in PBS
0.17
(pH 5.27
7.4) solution
1.07
as a function of time at 37 °C (Figure 9). In the case of SGF, it was observed that most of the drug,
SD means standard deviation; in order to obtain standard deviation, the experiment was performed in triplicate for
around each88%
film.from CTH-1 and 85% from CTH-2, was released in 50 min. On the contrary, in the PBS
solution, the CDR for CTH-1 and CTH-2 was around 93% in 90 min and 98% in 120 min, respectively.
Table 6. amount
The balance Data showing
of drug swelling
could not behaviour of CS/PAH
be determined becausefilms both
in CaCl
films2 solutions
converted of into
variable
gel-like
molar concentrations.
material due to acidic dissolution in SGF and fragmented in PBS after 30 min (CTH-1) and 120 min
(CTH-1). The drug CTH-0release in PBSCTH-1solution atCTH-2120 min for CTH-1 CTH-3 sample was significantlyCTH-5
CTH-4 higher in
[CaCl2 ]
the CTH-2 Swelling
sample (mean SD = 95.30%, SD
Swelling SD =Swelling
1.40) thanSDthe Swelling
CTH-1 sample SD (mean = 91.44%,
Swelling SD SD = 1.87),
Swelling SD t
(g/g)
(4) = −2.86, p-value < (±)
0.05. A(g/g)
similar(±)
pattern(g/g) (±)
was observed (g/g)
for the (±) (g/g)
SGF solution; (±)
mean (g/g)
for (±)
the CTH-1
0.1 M 30.95 0.99 25.54 0.99 18.69 0.56 - - 24.83
sample was 88.39% (SD = 0.51), while the mean was 84.78% (SD = 0.48) for the CTH-2 sample; t (4) = 0.84 23.87 1.21
0.3 M 14.55 0.49 17.22 1.26 12.16 1.63 - - 8.66 1.06 13.96 0.74
8.87,0.5p-value
M <7.04
0.005. 0.90
From these
9.50 results,
1.59 it could
10.82 be 0.69
deduced 8.91that the
1.69drug 7.48
was released
0.15 in7.51
a controlled
0.56
0.7 M in the
manner 8.33 1.50
PBS solution, 10.38 1.59
when compared 9.06to the0.57
SGF. 8.10 0.01 6.76 0.37 10.40 1.63
0.9 M 5.67 0.98 10.06 0.97 9.18 1.89 7.09 0.75 8.82 0.18 11.70 0.62
1.0 M 7.11 0.25 11.88 1.07 7.80 0.68 4.42 0.26 11.17 2.86 8.15 1.23
SD means standard deviation; in order to obtain standard deviation, the experiment was repeated three times for
each film.

3.6. Cumulative Drug Release (CDR) Study

Ciprofloxacin hydrochloride monohydrate, a model drug, was loaded in two films coded as
CTH-1 and CTH-2 (Table 7). Based on encouraging results as described in Sections 3.1–3.5, the CTH-1
and CTH-2 blend films were selected. These two blend films had a greater concentration of natural
polymer used in the present study (chitosan). However, CTH-3 and CTH-5 showed more solubility in
aqueous media and CTH-4 film had a greater concentration of PAH. The controlled drug release from
both films (CTH-1 and CTH-2) was studied in SGF (pH 1.2) as well as in PBS (pH 7.4) solution as a
function of time at 37 ◦ C (Figure 9). In the case of SGF, it was observed that most of the drug, around
88% from CTH-1 and 85% from CTH-2, was released in 50 min. On the contrary, in the PBS solution,
the CDR for CTH-1 and CTH-2 was around 93% in 90 min and 98% in 120 min, respectively. The
Pharmaceutics 2020, 12, 131 13 of 18

balance amount of drug could not be determined because both films converted into gel-like material
due to acidic dissolution in SGF and fragmented in PBS after 30 min (CTH-1) and 120 min (CTH-1).
The drug release in PBS solution at 120 min for CTH-1 sample was significantly higher in the CTH-2
sample (mean = 95.30%, SD = 1.40) than the CTH-1 sample (mean = 91.44%, SD = 1.87), t (4) = −2.86,
p-value < 0.05. A similar pattern was observed for the SGF solution; mean for the CTH-1 sample was
88.39% (SD = 0.51), while the mean was 84.78% (SD = 0.48) for the CTH-2 sample; t (4) = 8.87, p-value
< 0.005. From these results, it could be deduced that the drug was released in a controlled manner in
Pharmaceutics 2020, 12, x 13 of 18
the PBS solution, when compared to the SGF.
Table 7. Data on cumulative drug release (%) in SGF and PBS solutions.
Table 7. Data on cumulative drug release (%) in SGF and PBS solutions.
CTH-1 * SD CTH-1 * SD CTH-2 SD CTH-2 SD
Time
Time(min) CTH-1 CTH-1
(PBS)% * SD (±)(±) (SGF)% * SD (±)(±) CTH-2 (PBS)% SD(±) (±)
CTH-2
(SGF)% SD (±) (±)
(min)0 (PBS)%
0.00 0.00 (SGF)% 0.00 0.00 (PBS)% 0.00 0.00 (SGF)%
0.00 0.00
010 0.00
62.11 0.00
0.90 0.00
40.21 0.00 0.30 0.0046.47 0.00
0.50 0.00
47.21 0.000.30
1020 62.11
68.19 0.90
1.00 40.21
66.42 0.30 0.50 46.47 64.95 0.50
0.60 47.21
56.56 0.300.50
2030 68.19
78.55 1.00
1.20 66.42
80.45 0.50 0.30 64.95 75.60 0.60
0.40 56.56
80.45 0.500.30
3040 78.55
90.50 1.20
1.50 80.45
88.26 0.30 0.30 75.60 79.88 0.40
0.70 80.45
84.74 0.300.30
4050 90.50
91.30 1.50
0.90 88.26
88.39 0.30 0.60 79.88 84.63 0.70
0.10 84.74
84.80 0.300.60
50 91.30 0.90 88.39 0.60 84.63 0.10 84.80 0.60
60 92.73 2.00 -- -- 89.59 0.30 -- --
60 92.73 2.00 – – 89.59 0.30 – –
70 89.90 2.00 -- -- 89.99 0.50 -- --
70 89.90 2.00 – – 89.99 0.50 – –
80 80 91.46
91.46 1.80
1.80 – -- – -- 90.19 90.19 0.30
0.30 – -- – --
90 90 92.09
92.09 1.80
1.80 – -- – -- 91.64 91.64 0.30
0.30 – -- – --
100100 91.49
91.49 1.60
1.60 – -- – -- 94.49 94.49 0.60
0.60 – -- – --
110110 91.13
91.13 1.20
1.20 – -- – -- 97.91 97.91 0.20
0.20 – -- – --
120120 91.44
91.44 0.60
0.60 – -- – -- 95.31 95.31 0.60
0.60 – -- – --
** SD
SDmeans
meansstandard
standard deviation;
deviation; in order
in order to obtain
to obtain standardstandard
deviation,deviation, the experiment
the experiment was repeatedwas
threerepeated
times for
each
threefilm.
times for each film.

Figure 9. Data on cumulative drug release (%) in SGF and PBS solutions.
Figure 9. Data on cumulative drug release (%) in SGF and PBS solutions.
4. Conclusions
4. Conclusions
In this study, a drug delivery matrix based on chitosan/PAH blend films with different compositions
In this study,
was successfully a drugbydelivery
prepared matrix
the solution based
casting on chitosan/PAH
method blend films with
and their physicochemical different
characteristics
compositions
were evaluated.wasThese
successfully prepared
films were foundbyto the
havesolution casting method
a homogenous and their
architecture physicochemical
and excellent surface
characteristicsAFM
morphology. wereexhibited
evaluated. These filmsinwere
improvements found
surface to have when
morphology a homogenous architecture was
the PAH concentration and
excellent surface morphology. AFM exhibited improvements in surface morphology when the PAH
concentration was increased up to 60% (w/w). CTH-1 (90% chitosan and 10% PAH) showed maximum
WCA, which was 115°. Moreover, the CS/PAH blend films exhibited varied swelling responses in
different media and sustained drug release behaviour in PBS, as compared to SGF. The swelling
response of blend films in deionized water showed a linear increase as a function of time, and CTH-
Pharmaceutics 2020, 12, 131 14 of 18

increased up to 60% (w/w). CTH-1 (90% chitosan and 10% PAH) showed maximum WCA, which was
115◦ . Moreover, the CS/PAH blend films exhibited varied swelling responses in different media and
sustained drug release behaviour in PBS, as compared to SGF. The swelling response of blend films in
deionized water showed a linear increase as a function of time, and CTH-1 (90% CS and 10% PAH) had
a higher degree of swelling, which was around 122 g/g after 80 min, as compared to the other blend
films. The maximum observed swelling value in NaCl (0.1 M) for CTH-1 was 41 g/g and in CaCl2 (0.1
M) 31 g/g for CTH-0 (100% CS). Two films were selected, CTH-1 (90% CS and 10% PAH) and CTH-2
(80% CS and 20% PAH), for in vitro CDR at 37 ◦ C. In SGF (pH 1.2), the CDR for CTH-1 and CTH-2 was
about 88% and 85% in 50 min, respectively. In SGF, both films were converted into gel-like material
after 30 min due to lesser stability of films at lower pH. In the PBS solution (pH 7.4), meanwhile, the
CDR for CTH-1 and CTH-2 was around 93% in 90 min and 98% in 120 min, respectively. It can be
concluded that the drug was released in a controlled manner in the PBS solution, as compared to the
SGF. The selected blend films showed a relatively reduced release (CHT1 = 88% in 50 min, CTH2 = 85%
50 min) in simulated gastric fluid (pH = 1.2) and maximum drug release (CHT1 = 93% in 90 min, CTH2
= 98% 120 min) in phosphate buffer saline (pH = 7.4), thus indicating a pH-responsive (smart) nature.
Although the CTH-1 and CTH-2 films showed remarkable drug release, they converted to a gel-like
material over a period of time (30 min) in acidic pH (pH = 1.2). This showed the pH-responsive nature
of CTH-1 and CTH2. In summary, the results show that chitosan CS/PAH are excellent drug delivery
matrices for drug release at pH 7.4. These blend films can be employed for injectable drug delivery
systems, tissue regeneration, and associated biomedical applications such as wound dressing due to
good compatibility between the drug and the film matrix; they may, however, may not be suitable
candidates for oral drug administration.

Author Contributions: Conceptualization, A.G. and M.S.S.; methodology, M.S.S., Q.H., and M.A.A.; validation,
M.S.S., and Q.H.; formal analysis, M.L., Z.K., and M.S.Z.; data curation, A.G., M.S.S., M.A.A., and Z.K.;
writing-original draft preparation, A.G. and M.L.; writing-review and editing, A.G., M.L., M.S.Z., and Z.K. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors are thankful to the Higher Education Commission of Pakistan for giving M.S.S.
the opportunity to conduct this research project at the Department of Food Science, Rutgers, The State University
of New Jersey USA.
Conflicts of Interest: The authors declare no conflict of interest.

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