Journal of Drug Delivery Science and Technology 67 (2022) 102952

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Journal of Drug Delivery Science and Technology

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Opinion Paper

β-cyclodextrin modification by cross-linking polymerization as highly

porous nanomatrices for olanzapine solubility improvement; synthesis,
characterization and bio-compatibility evaluation
Kifayat Ullah Khan a, Muhammad Usman Minhas b, *, Syed Faisal Badshah a, Muhammad Sohail c,
Rai Muhammad Sarfraz b
a
Department of Pharmaceutics, Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur, 63100, Punjab, Pakistan
b
College of Pharmacy, University of Sargodha, University Road, Sargodha City, Punjab, Pakistan
c
Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad, 22060, KPK, Pakistan

A R T I C L E I N F O A B S T R A C T

Keywords: Herein, we report highly porous and amorphous nanomatrices developed by cross-linking polymerization with
Porous nanomatrices subsequent condensation technique for solubility, dissolution and ultimately bioavailability enhancement of
β-cyclodextrin (β-CD) poorly soluble drug olanzapine (OLN). β-cyclodextrin was chemically cross-linked with monomer 2-acrylamido
Solubility enhancement
2-methylpropane sulfonic acid (AMPS) by using methylene bis acrylamide (MBA) as cross-linking agent.
Cross-linking polymerization
Olanzapine
Developed nanomatrices were characterized by zetasizer, FTIR, XRD, SEM, DSC, swelling, sol-gel, drug loading,
In-vitro & in-vivo evaluation stability, porosity (%), solubility, and in-vitro dissolution analysis. The developed porous nanomatrices are
intended for oral administration of poorly soluble drugs therefore, to determine the biocompatibility of the
system to the biological environment, in-vivo cytotoxicity study was also conducted using rabbit model which
endorsed the safety of the nanomatrices with biological system. The particle size of nanomatrices were found
152.30 ± 07.46 d.nm. XRD analysis depicted the highly amorphous nature of nanomatrices while. To evaluate
the pharmacokinetic profile of olanzapine, in-vivo studies were also conducted. The in-vivo and dissolution ex­
periments revealed that drug release characteristics were significantly improved by nanomatrices as compared
with the reference product (OLANZIA®). The solubility of olanzapine by the developed nanomatrices was
enhanced noticeably up to 40.11 times which is higher than previously reported β-cyclodextrin formulations. The
efficient method of preparation, improved solubility, rapid and high dissolution and non-toxic βCD-co-poly
(AMPS) nanomatrices may be a promising approach for oral delivery of poorly soluble drugs.

1. Introduction structure of nanomatrices has a capacity to entrap active entities as well

Nanomatrices presents a promising approach to enhance the solu­
bility, release characteristics, and bioavailability of poorly soluble drugs
due to their small sizes, large surface area, high amorphous nature, soft
biomaterial, high swelling and drug loading capacity, porous structure,
bio-compatibility, and minimal resources required for its
manufacturing. Nanomatrices are solvent swollen nano version of
hydrogels with added qualities of nano-systems [1,2]. It may be defined
as highly cross-linked three dimensional nano sized hydrogels with size
range of 20–200 nm [3]. Both hydrophilic and hydrophobic drugs can be
loaded in the nanomatrices and can be administered by oral, nasal,
topical, and parenteral route [4–7]. The spongy and highly porous
as to modulate the release pattern.
Poor aqueous solubility is one of the major issue of new chemical
entities that leads to low bioavailability, reduced therapeutic effects and
dosage escalation [8,9]. Poor soluble drugs leave gastro-intestinal tract
(GIT) prior their dissolution which reduce their clinical efficacy. By
some estimation, about 90% of new chemical entities and 40% of the
marketed products are suffered from poor aqueous solubility [10,11].
There is a mounting need of exploring commercially practicable and
economical strategies for solubility enhancement of poorly soluble
drugs. To tackle the issue, different strategies have been explored by the
researchers i.e. A Naqvi and M Imono et al. have prepared nanocrystals
[12,13], Morina Deniz et al. cyclodextrins complexes [14], Minhas and

* Corresponding author.
E-mail addresses: us.minhas@hotmail.com, usman.minhas@uos.edu.pk (M.U. Minhas).

https://doi.org/10.1016/j.jddst.2021.102952
Received 9 July 2021; Received in revised form 26 September 2021; Accepted 27 October 2021
Available online 8 November 2021
1773-2247/© 2021 Elsevier B.V. All rights reserved.
K.U. Khan et al. Journal of Drug Delivery Science and Technology 67 (2022) 102952

Table 1 drug side effects such as; gastric or ocular irritation [39–41]. Amongst
Feed ratio of β-cyclodextrin, monomer (AMPS), cross-linking agent (MBA), other natural cyclodextrins, β-CD is relatively safer, perfect cavity size,
initiator (APS), and %drug loaded content in nanomatrices formulations. efficient drug complexation and loading, low cost, and has been the most
S/ Formulation β-CD AMPS MBA APS %DLCa studied cyclodextrin [42,43].
N Code (g) (g) (g) (g) Olanzapine (OLN) is selected as a model drug which is one of the
01 BNG-1 0.5 02 02 0.2 79.78 ± most relevant second generation atypical antipsychotic drug used in the
0.99 treatment of schizophrenia, acute mania with bipolar disorder, depres­
02 BNG-2 01 02 02 0.2 84.14 ± sion, agitation, and psychotic symptoms in dementia [44–47]. OLN,
1.12
chemically known as 2-methyl-4-(4-methyle-1-piperazinyl)-10H-thieno
03 BNG-3 1.5 02 02 0.2 86.06 ±
1.45 (2, 3 b) (1,5) benzodiazepine. It is approved by US Food and Drug
04 BNG-4 1.5 04 02 0.2 88.82 ± Administration (FDA) and is among the top 20 prescription drug
1.43 [48–51]. It bind and antagonize many receptors including dopamine 1,
05 BNG-5 1.5 06 02 0.2 93.55 ± 2, 3, and 4, 5HT3, 5HT2A, 5HT2C, 5HT6, histamine at H1 receptors, and
2.86
06 BNG-6 1.5 02 04 0.2 76.04 ±
alpha-1 adrenergic receptors [52,53]. Despite of its vast applications in
2.55 treating psychotic disorders, OLN is a poorly soluble drug (43 mg. L− 1)
07 BNG-7 1.5 02 06 0.2 69.94 ± belong to BCS class-II which is an obstacle to the schizophrenia treat­
1.91. ment [49,54]. Therefore, it is vital to develop an efficient pharmaceu­
a
Values are calculated as mean ± SD (n = 3). tical dosage form which allows better pharmacokinetic profile of
olanzapine.
Over the past few decades, psychotic disorders specifically schizo­
phrenia and depression has dramatically increased affecting 1% of the
world population [55]. Among numerous challenging mental health
disorders, schizophrenia is chronic, and one of the most severe and
debilitating mental disorder [56]. The symptoms include hallucinations,
delusions, suicidal tendency, thought/movement disorders, trouble in
focusing attention and usually complete lack of emotions. By some
estimation, 50% of the schizophrenia patients are suffered from
depression and is one of the most leading cause of suicide worldwide
[57]. Schizophrenia and bipolar mental disorders can be effectively
treated with olanzapine as monotherapy [58].
In current study, nanomatrices drug delivery system has been
explored for solubility, bioavailability, and release characteristics
improvement of poorly soluble drug olanzapine (OLZ). The β-CD-co-
poly (AMPS) based nanomatrices are developed by cross-linking poly­
merization with subsequent condensation technique that would
demonstrate a potential advantage over the previously reported systems
in terms of solubility, dissolution, and bioavailability.

2. Materials and methods

2.1. Chemicals
Fig. 1(a). Physical appearance of developed nanomatrices.

Olanzapine (OLN) (molecular weight: 312.4 g/mol) API was ob­

Qandeel Khalid et al. nanogels and nano-sponges respectively [15,16] Q
Fu and J Zu et al. salt formation [17,18], Thambe and X She et al. pre­
pared amorphous solid dispersions [19,20], Rimpy Diwan, Y Chen and
N. Baolong et al. nanoparticles [21–23], Y Ikeuchi-Takahashi et al.
micro-particles [24], Y Bi et al. liposomes [25], A Alshweiat and Q Liu
et al. nano-suspension [26], M Gao et al. nano-capsule [27], X Shi et al.
solid amorphous dispersions [19], J liang and C Sun et al. micelles [28,
29], J Mao et al. polymeric microneedles [30], F Najafi et al. dendrimers
[31], and S Potharaju et al. used self-emulsifying drug delivery system
(SEDDS) for solubility enhancement of poorly soluble drugs [32].
For nano drug delivery systems, intended for solubility enhance­
ment, hydrophilic excipients seem to be the most up-front and promising
solution. For this purpose, β-cyclodextrin (β-CD) and AMPS are used in
current study as a fundamental excipient for the synthesis of nano­
matrices. β-CD is a hydrophilic cyclic oligosaccharide having relatively
hydrophobic central cavity and outer hydrophilic surface. Via this
structure, β-CD is capable of encapsulating and enhancing the aqueous
solubility and ultimately the bioavailability of poorly soluble drugs with
in a drug release system [33–35]. Many researchers have reported
β-cyclodextrin mediated solubilization of poorly soluble drugs [36–38].
In the pharmaceutical industry, cyclodextrins especially beta ones have
been mainly used to enhance the aqueous solubility, bio-availability,
and stability of poorly soluble drugs. Moreover, it can also reduce
tained from the Global Pharmaceutical Pakistan, β-cyclodextrin (β-CD)
(molecular weight: 1135 g/mol), monomer 2-acrylamido 2-methyl­
propane sulfonic acid (AMPS) (molecular weight: 207.24 g/mol), cross-
linking agent methylenebisacrylamide (MBA) (molecular weight:
154.169 g/mol), initiator ammonium persulfate (APS) (molecular
weight: 228.18 g/mol) were purchased from Sigma Aldrich. Other
chemicals used in study were of reagent grade.
2.2. Methods
2.2.1. Synthesis of nanomatrices
Cross-linking polymerization with slight modifications of our previ­
ously reported technique was used for the synthesis of nanomatrices [15,
59]. Clear solutions of β-CD, 2-acrylamido-2-methylpropane sulfonic
acid (AMPS), and methylene bis-acrylamide (MBA) were prepared
separately by using magnetic stirrer. After this, weighed amount of
ammonium persulfate (APS) was added to the previously prepared
beta-cyclodextrin solution with continuous stirring. Finally, β-CD/APS
mixture, AMPS and MBA solutions were combined in round bottom
flask. In order to complete the polymerization reaction, reflux conden­
sation was carried out for 4–6 h until solvent was distilled out at
75–85 ◦ C. After this, fluffy gels were collected, sieved and dried in oven
at 40 ◦ C for 24 h. Different ingredients used in nanomatrices

2
K.U. Khan et al.
Fig. 1(b). Proposed schematic diagram of β-CD based nanomatrices.
formulations are shown in Table 1, while physical appearance and
proposed schematic diagram of developed nanomatrices is presented in
Fig. 1 (a & b) respectively.
2.2.2. Drug loading of nanomatrices
Swelling diffusion method was adopted for loading of drug (OLN)
into naïve nanomatrices formulations. For this purpose, 2% olanzapine
(OLN) solution w/v was prepared in solvent mixture of methanol and
distilled water (4:1). Then weighed quantity (500 mg) of prepared
nanomatrices was added into the OLN solution and sonicated for 30–40
min. After sonication, the whole mixture (OLN/nanomatrices) was kept
overnight for swelling purpose at 25 ◦ C ± 01. After this, the mixture was
lyophilized for 4–6 h and characterized in-vitro & in-vivo.
3. IN-VITRO characterization of nanomatrices
3.1. Particle size analysis
It is well known that particle size determination is very important
while working on solubilization enhancement of poorly soluble drugs as
particle size is directly related to the solubility. Size analysis of prepared
nanomatrices formulation was carried out using zeta sizer (Malvern Zeta
Size Nano ZS, UK). Samples were suspended for analysis with filtered
(pore size 0.22 μm) ultra-pure water [60,61]. The analysis was done in
triplicate.
3.2. FTIR analysis
To determine different functional groups and to analyze the complex
network of developed nanomatrices, fourier transform infrared spec­
troscopy (FTIR) was performed for pure drug olanzapine (OLN),
β-cyclodextrin (β-CD), monomer AMPS, and OLN-loaded nanomatrices
formulation using FTIR spectrophotometer (Tensor 27 series, Bruker Co.
Germany). Before analysis, all samples were passed through mesh 60
and placed on the ATR cell in specific quantity. IR spectrum of each
sample was scanned in the range of 4000–600 cm− 1 at resolution of 4
cm− 1.
3.3. Scanning electron microscopy (SEM)
SEM analysis was performed to visualize the surface morphology of
prepared nanomatrices formulations using scanning electron micro­
scope (JSM-6490A, Tokyo, Japan). Before analysis, nanomatrices sam­
ples were gold platted for better conductivity, and then glued on
aluminum stub using double adhesive carbon tape. The micrographs
were captured at different magnifications.
3.4. X-ray diffraction (XRD) analysis
XRD analysis was carried out to monitor the crystalline structure of
olanzapine (OLN) before and after incorporation into the prepared
nanomatrices drug delivery system. For this purpose, powder samples of
pure drug OLN, blank and OLN-loaded nanomatrices were analyzed
3
Journal of Drug Delivery Science and Technology 67 (2022) 102952
using x-ray diffractometer (D8 Discover, Bruker, Germany). The data
was collected in the range of 0-60 ◦ C at 2Ɵ.
3.5. Differential scanning calorimeter (DSC) analysis
For thermal analysis of the developed nanomatrices, differential
scanning calorimeter (DSC) (Q600 series TA, USA) analysis was carried
out for pure βeta-cyclodextrin (β-CD), 2-acrylamido-2-methyle propane
sulfonic acid (AMPS), and developed nanomatrices. DSC analysis was
performed by placing specific quantity (0.5–3 mg) of samples in stan­
dard aluminum pan. Temperature range was kept at 25-600 ◦ C and heat
rate was adjusted at 20 ◦ C/min under nitrogen flow rate of 10 mL/min.
All samples were analyzed in triplicate.
4. IN-VITRO studies of nanomatrices
4.1. Solubility studies
Solubility study/test was performed to determine the solubility
enhancement of olanzapine (OLN) by the developed nanomatrices. For
this purpose, excess amount of pure drug OLN and prepared nano­
matrices formulations (BNG-1 to BNG-7) equivalent to 100 mg of drug
were dispersed separately in pre-labeled containers containing distilled
water, HCl solution of pH 1.2, and phosphate buffer of pH 6.8 using
magnetic stirrer at 200 rpm overnight at room temperature. The nano­
matrices solutions were added to respective drug solutions with
continuous stirring. After this, solutions were filtered and analyzed for
absorbance at lambda max 228 nm using UV-spectrophotometer [62].
4.2. Swelling studies
Swelling studies were performed to analyze the swelling behavior of
naïve nanomatrices formulations. A reported tea bag test was adopted
for swelling studies, for this purpose, specific quantity of prepared
nanomatrices (100 mg) was weighed and enclosed in empty tea bags
which were then immersed in pre-determined containers of respective
buffer solutions of pH 1.2 and 6.8 at 37 ◦ C ± 1. At pre-determined time
intervals of 2, 5, 10, 15, 20, 30, 40, 50, 60, 90, 120, 150, and 180 min,
the nanomatrices samples were taken out and blotted with filter paper
and hanged till no drop was oozing out from the bags and carefully
weighed and returned to its respective container. The swelling index of
nanomatrices formulations were calculated using equation (1).
Swelling index (Q) = W2/W1 1
Where W2 and W1 indicate the weight of swollen and dried nano­
matrices sample respectively.
4.3. Percent drug loaded content and entrapment efficiency (%DLC/EE)
Extraction technique was used for the assessment of percent drug
loaded content (%DCL) and entrapment efficiency (%EE) of the pre­
pared nanomatrices formulations. The %EE was done to estimate the
productivity of nanomatrices preparation technique, for this purpose,
100 mg OLN-loaded nanomatrices formulations were weighed and
dispersed in 50 mL solution of phosphate buffer of pH 6.8 and allowed to
stir at 200 rpm for 1 h. After this, the mixture was filtered by membrane
filter of pore size 0.45 μm and analyzed by UV-spectrophotometer at
lambda max 228 nm. Equations (2) and (3) was used for calculating %
DLC and %EE. The experiment was performed in triplicate (n = 3) and
given as the mean ± standard deviation (SD).
Entrapped ​ drug ​ in ​ nanomatrices
Drug loaded content (%) = × 100 2
Weight of ​ nanomatrices
K.U. Khan et al.
Actual ​ drug ​ contents ​ in ​ nanomatrices
Drug entrapment efficiency (%) =
Theoretical drug contents in ​ nanomatrices
4.4. Sol-gel fraction
Sol-gel analysis was performed to determine the percent conversion
of reactants into product i.e., to measure the un-crosslinked reactants
(β-CD & AMPS) in the developed nanomatrices structure. Soxhlet
extraction method was adopted for sol-gel analysis, for this purpose,
specific quantity of dried nanomatrices (500 mg) W1 were taken in
round bottom flask containing distilled water and subjected to Soxhlet
extraction process for 5–6 h after this, nanomatrices were placed in
electric oven and allowed to dry at 40 ◦ C. After drying samples were
weighed again W2 to calculate the percent sol-gel fraction using equa­
tions (4) and (5).
(W1 –W2 )
% ​ Sol Fraction = × 100
W1
% ​ Gel Fraction = 100 − Sol ​ Fraction
W1 is the initial dry weight of nanomatrices before extraction while
W2 is the final dry weight of nanomatrices after extraction.
4.5. Stability and percent porosity studies of nanomatrices
Stability studies of olanzapine (OLN) loaded nanomatrices were
carried out according to ICH guidelines [63]. β-CD-co-poly (AMPS)
nanomatrices were filled in glass vials and kept in stability chamber
(Memmert Beschickung, Japan) at 40 ± 02 ◦ C with 75 ± 5% RH. Sam­
ples were analyzed for physical changes, percent drug loaded content (%
DLC), FTIR and solubilization efficiency at time period of 0, 3rd and 6th
month.
In order to estimate the porosity (%) of nanomatrices formulation,
solvent replacement technique was employed. Initially, dried formula­
tions were weighted and then immersed in distilled water, permit to
saturate and weighted. Saturated formulations were blotted and weight
variation was calculated accordingly. The porosity (%) was estimated by
following equation.
(Wh ​ − ​ Wd)
Porosity (%) = × 100
ρV
where, Wd = weight of dried formulation, Wh = weight of saturated
formulation, ρ = density of distilled water, and V = volume of respective
formulation.
4.6. In-vitro dissolution studies
In-vitro dissolution study was performed to determine the drug
release potential of β-cyclodextrin (β-CD) based nanomatrices and to
compare its release characteristics with the reference product OLAN­
ZIA®. Dissolution study was carried out in a calibrated six station
dissolution test apparatus (Curio, Pakistan) equipped with paddles using
500 mL medium of phosphate buffer of pH 6.8 and HCl solution of pH
1.2. Reported tea bag method was adopted for dissolution experiment
[64,65]. Specified quantity of nanomatrices samples were placed in tea
bags and immersed into respective dissolution medium. Paddle’s speed
and temperature of medium was set at 50 rpm and 37 ◦ C ± 0.2
respectively throughout experiment. At pre-determined time intervals,
5 mL aliquot was withdrawn and replaced with equal fresh dissolution
medium. To quantify the percent OLN release, withdrawn samples were
analyzed at lambda max 228 nm using UV-spectrophotometer.
Journal of Drug Delivery Science and Technology 67 (2022) 102952
× 100 3
5. In-vivo analysis
5.1. In-vivo cyto-toxicity study
In order to determine the safety and bio-compatibility of the devel­
oped nanomatrices to the biological system, toxicity study was con­
ducted on healthy male rabbits. The study protocols were reviewed and
granted under reference number 14/2020/PAEC by the institutional
committee i.e., Pharmacy Animal Ethics Committee (PAEC), Faculty of
Pharmacy, Department of Pharmaceutics, The Islamia University of
Bahawalpur (IUB) Pakistan. Twelve (12) healthy rabbits of 2.5 ± 0.3 kg
average body weight were procured and divided into two groups (six
rabbits in each group). Group-A was contemplated as control and group-
4 B was treated with prepared nanomatrices. 5 g/kg blank nanomatrices
was administered to test animals orally. Both groups were keenly
5 observed physically for water and food intake, salivation, tremors, uri­
nation, diarrhea, illness, response behavior, any irritation (ocular/
dermal), and mortality. After 15th day blood samples of both groups
were collected for the evaluation of blood biochemistry. Animals were
then sacrificed by slaughtering for histopathological examination of
vital organs (heart, liver, lungs, kidneys, stomach, spleen, and colon)
[66,67].
5.2. In-vivo high-performance liquid chromatography (HPLC) study
5.2.1. Chromatographic condition for olanzapine (OLN)
Olanzapine (OLN) was analyze in-vivo by already reported method
with slight modification [68]. ODS hypersil C18 (250 * 4.6 mm, Agilent
technologies, Netherland) stainless steel column was used. Plasma
sample was analyzed on an isocratic chromatographic system (Agilent
technologies, series 1200) consisted a pump and wavelength detector.
Acetonitrile and phosphate buffer of pH 6 (60:40 v/v) was used as
mobile phase and its pH was adjusted at 3.2 with the help of per-chloric
acid. Sonication was carried out in order to degas the mobile phase
followed by filtration using 0.45 μm Millipore filters (Merck, Germany).
Flow rate for mobile phase was 1 mL/min and 20 μL sample were
injected with a run time of 10 min. Calibration curve was formed with
6
concentration 25–1000 ng/mL.
5.2.2. Animals used for in-vivo studies
Twenty-four (24) healthy rabbits were procured for in-vivo studies
weighing 2.5 ± 0.2 kg. The study protocols were reviewed and granted
under reference number 14/2020/PAEC by the institutional committee
i.e., Pharmacy Animal Ethics Committee (PAEC), Faculty of Pharmacy,
Department of Pharmaceutics, The Islamia University of Bahawalpur
(IUB) Pakistan.
5.2.3. Study design
Animals were divided into two groups (twelve rabbits in each group)
i.e., group-A, and group-B. Single dose study design was carried out on
test animals. All animals (rabbits) were properly marked for identifica­
tion purpose and housed in ventilated steel cages with free access to
water and food. Group-A was contemplated as control and group-B was
treated with nanomatrices. Group-A (control) was administered 714 μg
of olanzapine (OLN) orally filled in hard gelatin capsule. Group-B was
administered loaded β-cyclodextrin (β-CD) based nanomatrices formu­
lation containing 714 μg drug olanzapine (drug calculated based on
human dose of 20 mg/day).
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K.U. Khan et al. Journal of Drug Delivery Science and Technology 67 (2022) 102952

Fig. 2. Particle size analysis of nanomatrices.

Fig. 3. FTIR spectra of (a) olanzapine (OLN), (b) β-cyclodextrin, (c) AMPS, (d) OLN-loaded nanomatrices.

5.2.4. Evaluation of pharmacokinetic parameters 6. Results and discussion

All the pharmacokinetic parameters i.e., Cmax, Tmax, Ke, Clast,
Tlast, AUC0-t, AUC0-∞, AUMC0-∞, t1/2, and mean plasma drug con­
centration were carefully estimated through PK-Solver add in program
and Microsoft Excel (2013). Plasma drug OLZ concentration in all
groups was estimated by means of calibration curve.
6.1. Particle size analysis
While working on solubility enhancement of poorly soluble drugs,
particle size analysis is an essential parameter in characterization
because particle size has direct role in solubilization enhancement. Ac­
cording to Ostwald Freundlich equation, solubility is directly

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K.U. Khan et al. Journal of Drug Delivery Science and Technology 67 (2022) 102952

Fig. 4. SEM micrographs of β-CD based nanomatrices at different magnifications.

proportional to particle size, i.e., solubility is increased when the par­

ticle size decreased vice versa [69]. Improved solubility, dissolution, and
bioavailability is an important characteristic of Nano systems. Zeta sizer
(Malvern Zeta sizer Nano ZS, UK) was used for particle size analysis and
observed that about 86% of nanomatrices particle were in range of
152.30 ± 07.46 nm with polydispersity index value (PDI) of 0.133
which indicated that nanomatrices have very low affinity to form clus­
ters or aggregates as shown in Fig. 2. It is well known that
nano-technology especially soft nanoparticles hugely influence the
biopharmaceutical performance of the system as reduced particle size
and higher surface area favors enhance dissolution/solubility and
bioavailability [2].

6.2. Structural analysis of nanomatrices

Fig. 3 presents the FTIR analysis of pure drug olanzapine (OLN),

β-cyclodextrin (β-CD), monomer 2-acrylamido-2-methylpropane sul­
fonic acid (AMPS), and OLN-loaded nanomatrices formulations. FTIR
analysis was carried out in a range of 4000–600 cm− 1 to ensure cross-
linking of β-cyclodextrin (β-CD) and APMS and to determine different
functional groups. FTIR spectrum of pure drug OLN showed character­
istics peaks at 3265 cm− 1 due to -O-H and -N-H stretching, 1593.98
cm− 1 due to –C–– C-, 1391.72 cm− 1 due to –C– – N-, absorption peak at
2960.34 cm was detected due to -C-H, and 755.88 cm− 1 due to -C-S-
− 1

stretching as presented in Fig. 3 (a). The infrared spectrum of β-cyclo­

Fig. 5. XRD patterns of (a) olanzapine (OLN), (b) blank nanomatrices, and (c)
OLN-loaded nanomatrices.
dextrin (β-CD) exhibited principle broad transmittance peak at 3100-
3300 cm− 1 due to the alcoholic stretching (-O-H) of primary and sec­
ondary hydroxyl groups linked by inter-molecular and intra-molecular
hydrogen bonding. Absorption peak at 2923.09 cm− 1 was due to the
-C-H stretching vibration of methyl (-CH3) and methylene (-CH2). Peak
at 1031.78 cm− 1 characterizes C– – O stretching vibration while asym­
metric absorption vibrations of C–O was observed at 1654.34 cm− 1 [70]
as shown in Fig. 3 (b). AMPS spectrum was recorded which showed
characteristic bands at 1233.92 cm− 1 and 1371.74 cm− 1 indicating

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K.U. Khan et al. Journal of Drug Delivery Science and Technology 67 (2022) 102952

asymmetric and symmetric stretching of S– – O respectively that

Fig. 6. DSC analysis of (a) pure βeta-cyclodextrin (β-CD), (b) AMPS and (c)
developed nanomatrices.
confirmed the SO3H group of AMPS. Principal peaks at 1658.59 cm− 1
and 1615.23 cm− 1 were due to C– – O stretching of amide-I and N–H
bending of amide-II respectively. Peak at 2910.81 cm− 1 was assigned to
-C-H stretching of CH2 group as shown in Fig. 3 (c).
FTIR spectrum of OLN-loaded nanomatrices formulation indicated
the successful cross-linking of β-cyclodextrin (β-CD) and monomer 2-
acrylamido-2-methylpropane sulfonic acid (AMPS) to form the com­
plex network, as all characteristics peaks of β-CD and monomer (AMPS)
were present with slight shifting of their frequencies as shown in Fig. 3
(c). Principal peaks of olanzapine (OLN) present in OLN-loaded nano­
matrices spectrum indicated the entrapment of drug in the amorphous
β-CD-co-poly (AMPS) nanomatrices network as shown in Fig. 3 (d).
The surface and cross-sectional micrographs of the prepared nano­
matrices are presented in Fig. 4. Scanning electron microscope (SEM)
was used to evaluate the surface morphology of nanomatrices at
different magnifications. From SEM cross-sectional views, nanomatrices
were found spongy and highly porous. The numerous pores present in
the system act as solvent entry channels which favors release of drug
from the system upon contact with aqueous medium.
Fig. 5 (a, b, and c) presented x-ray diffraction (XRD) patterns of pure
drug olanzapine (OLN), blank nanomatrices, and OLN-loaded nano­
matrices formulations respectively. XRD analysis is one of the most
needed parameters to be considered while working on solubility
enhancement of poorly soluble drugs. Crystalline drugs have poor sol­
ubility as compared to drugs with amorphous nature. Amorphous form
is directly related to enhanced solubility, higher absorption/dissolution
rate, and increase bioavailability of poorly soluble drugs [71]. By some
estimates, solubility of poor soluble drugs can be enhanced 10–1600
times by applying the amorphous form [72]. XRD analysis was carried
out to analyze the crystallinity of pure olanzapine and to analyze the
potential changes made in olanzapine nanocrystals after incorporating
into nanomatrices formulation.
The x-ray diffractogram of olanzapine (OLN) exhibited various
principal peaks at diffraction angel of 2 theta, in the range of 10 to 30◦ i.
e., 8.66, 11.02, 13.31, 15.42, 17.51, 19.84, 21.06, 22.23, 26.41 which
point out high crystallinity of OLN as shown in Fig. 5 (a). XRD pattern of
blank nanomatrices (Fig. b) shown that there was lack of any sharp peak
which confirmed the amorphous nature of prepared nanomatrices. In
contrast, in the XRD pattern of OLN-loaded nanomatrices, the sharp
diffraction peaks of olanzapine (OLN) persisted with distinct decrease in
their intensity compared with XRD pattern of pure OLN crystals as
shown in Fig. 5 (c). The lesser and broader peaks of OLN in loaded
nanomatrices pointed out that OLN was successfully incorporated in the
amorphous system of nanomatrices, comprised of hydrophilic (β-CD)
and monomer (AMPS), which imparted its features to OLN.

6.3. DSC analysis of developed nanomatrices

Fig. 6 presented the DSC thermograms of β-CD, AMPS and nano­

matrices. In DSC thermogram of pure β-CD first endothermic peak was
observed at 107 ◦ C and 2nd peak at 330 ◦ C which was due to moisture
loss and melting phase respectively. Previously, Malik et al. also re­
ported similar peaks of β-CD [73]. DSC thermogram of AMPS displayed a
characteristic sharp endothermic peak at 217 ◦ C which represented its
melting point as shown in Fig. 6. C Zhang et al. reported similar peaks of
AMPS [74]. The DSC thermogram of β-CD-co-poly (AMPS) nanomatrices
was different from the pure ingredients. The characteristic endothermic
peaks of β-CD at 107 ◦ C and that of AMPS at 217 ◦ C were completely
Fig. 7. Sol-gel analysis of β-CD based nanomatrices formulations (BNG-1 to disappeared in nanomatrices formulation which indicated the formation
BNG-7). of new polymeric network and thermal stability.

7
K.U. Khan et al. Journal of Drug Delivery Science and Technology 67 (2022) 102952

amount of uncross-linked β-cyclodextrin (β-CD) and monomer AMPS in

nanomatrices structure. Fig. 7 presented the sol-gel fraction of prepared
nanomatrices formulations (BNG-1 to BNG-2). The gel fraction was
significantly increased by increasing feed content of β-CD and monomer
AMPS as shown in Table 1. The trend may be attributed to the fact that
increase feed content of β-CD and monomer provided more active sites
(free radicals) during the polymerization reaction due to which more
stable gel was formed. Ali Muhammad et al. synthesized gelatin-
cyclodextrin hydrogels and found similar effect of β-CD on gel fraction
[75]. Another study based on the development of PVA-AA-AMPS
hydrogels found similar results of AMPS on gel fraction [76]. Similar
effect of β-CD and monomer on gel fraction are reported by Kaleem and
Yin. L., et al. [77,78]. In current study, the effect of cross-linking agent
MBA (methylene bis acrylamide) was also evaluated. It was found that
gel fraction was decreased by increasing MBA feed content (Table 1)

Fig. 8 (a). Swelling index of β-CD based nanomatrices formulations at HCl

solution of pH 1.2.
Table 2
Percent porosity (%) of β-cyclodextrin based nanomatrices.
S. No Formulation code Varying component Porositya (%)

01 BNG-1 β-CD 71.538 ± 0.701

02 BNG-2 76.021 ± 0.497
03 BNG-3 80.962 ± 0.893
04 BNG-4 AMPS 84.786 ± 0.990
05 BNG-5 89.381 ± 0.750
06 BNG-6 MBA 69.461 ± 1.002
07 BNG-7 64.792 ± 0.983
a
Values are calculated as mean ± SD (n = 3).

Table 3
Stability studies of olanzapine (OLN) loaded nanomatrices.
S. Parameters 0-month 3rd month 6th month
No

01 Physical appearance Initially No significant No significant

yellowish change change
color
Fig. 8 (b). Swelling index of β-CD based nanomatrices formulations at phos­ 02 Percent drug loaded 93.55 ± 2.86 92.974 ± 93.042 ±
phate buffer of pH 6.8. content (%DLC) 2.36 2.137
03 FT-IR analysis Performed No significant No significant
change change
6.4. Sol-gel fraction
04 Solubility Enhance up-to No significant No significant
enhancement of OLN 40.11 folds change change
Sol-gel fraction was determined by procedure mentioned in mate­ by nanomatrices
rials and methods for all blank nanomatrices formulations to analyze the

Fig. 9. Solubility of drug OLN in β-CD based nanomatrices (BNG-1 to BNG-7) and in pure form, in distilled water, HCl solution of pH 1.2, and phosphate buffer of
pH 6.8.

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K.U. Khan et al. Journal of Drug Delivery Science and Technology 67 (2022) 102952

Table 4
Clinical observations of control group-A and nanomatrices treated group-B.
CLINICAL OBSERVATIONS GROUP-A GROUP-B (treated with
(control) β-CD based nanomatrices)

BODY WEIGHT (kg) 1.94 ± 0.03 1.95 ± 0.05

Pretreatment 1.97 ± 0.04 1.96 ± 0.04
At day-7 1.98 ± 0.02 1.95 ± 0.02
At day-14
FOOD CONSUMPTION (g) 72.45 ± 1.03 71.88 ± 1.23
Pretreatment 74.11 ± 1.21 74.04 ± 1.22
At day-7 74.78 ± 1.33 73.98 ± 1.02
At day-14
WATER INTAKE (mL) 189.23 ± 187.32 ± 2.03
Pretreatment 1.32 190.45 ± 1.56
At day-7 190.03 ± 192.78 ± 2.33
At day-14 1.90
193.00 ±
1.87
SKIN TOXICITY (ulceration to skin, Normal Normal
redness, rashes, and baldness)
Fig. 10 (a). Dissolution profile of prepared nanomatrices and reference product OCULAR TOXICITY (redness, Normal Normal
at HCl solution of pH 1.2. lacrimation)
MORTALITY RATE Nil Nil

Values are calculated as mean ± SD (n = 3).

Table 5
Hematological analysis of both control group-A and treated group-B.
HEMATOLOGICAL GROUP-A GROUP-B (treated with β-CD based
ANALYSIS (control) nanomatrices)

Red blood cells count (106/ 5.78 ± 0.54 5.91 ± 0.33

mm3)
White blood cells count 6.21 ± 0.34 6.08 ± 0.62
(109/L)
Platelets count (109/L) 211.02 ± 1.32 203 ± 1.78
Hemoglobin (g/dL) 13.22 ± 0.45 13.34 ± 0.51
Monocytes (%) 03.87 ± 0.32 03.93 ± 0.12
Lymphocytes (%) 40.34 ± 1.01 41.02 ± 0.91
Neutrophils (%) 56.71 ± 2.00 53.86 ± 1.67
Eosinophil (%) 03.23 ± 0.45 03.76 ± 1.04

Values are calculated as mean ± SD (n = 3).

Fig. 10 (b). Dissolution profile of prepared nanomatrices and reference prod­

uct at phosphate buffer solution of pH 6.8.
Table 6
Bio-chemical analysis of Liver function of both control group-A and nano­
matrices treated group- B.
Biochemical analysis Group-A Group-B (treated with β-CD based
(control) nanomatrices)

Total cholesterol (mg. 63.34 ± 1.65 61.87 ± 2.04

dL− 1)
Total uric acid (mg. 03.55 ± 1.10 03.39 ± 1.43
dL− 1)
Total bilirubin (mg. 0.8 ± 0.98 0.9 ± 0.71
dL− 1)
Serum urea (mg.dL− 1) 15.23 ± 1.56 16.06 ± 1.77
Creatinine level (mg. 1.21 ± 0.34 0.90 ± 1.04
dL− 1)
Triglycerides level (mg. 53.94 ± 1.41 54.01 ± 1.88
dL− 1)
Fig. 10 (c). Drug release mechanism from loaded nanomatrices system. S.G.P.T (ALT) (IU.L− 1) 142.33 ± 2.04 145.21 ± 1.98
S.G.O.T (AST) (IU.L− 1) 28.71 ± 1.76 27.22 ± 2.59
Alkaline phosphate (IU. 47.90 ± 2.01 54.29 ± 1.33
although MBA is vital for cross-linking polymerzation as shown in Fig. 7. L− 1)
It is frequently reported in the literature that gel fraction increases by
Values are calculated as mean ± SD (n = 3).
increasing MBA concentration [79,80] but in the present study we found
decrease in gel fraction with increasing MBA concentration (BNG-6,
BNG-7) as shown in Fig. 7, which may be due to the formation of 6.5. Swelling study of nanomatrices
compact mass of reaction mixture because of unavailability of enough
primary free radicals (active sites) of β-CD and AMPS for Swelling of the system upon contact with aqueous medium is directly
polyemerization. related to drug release. Swelling study of prepared nanomatrices for­
mulations were conducted in pH condition of 1.2 (HCl solution) and 6.8
(phosphate buffer) to evaluate the effect of pH on nanomatrices swelling

9
K.U. Khan et al. Journal of Drug Delivery Science and Technology 67 (2022) 102952

Fig. 11. Histopathological analysis of vital organs i.e., control group-A and nanomatrices treated group-B.

behavior. All prepared nanomatrices formulations (BNG1-BNG7) had methylpropane sulfonic acid) that are protonated to –SO3H groups in
shown good swelling behavior at both pH condition but more in high pH acidic pH. The strong interaction between sulfonate groups due to
condition (pH 6.8) as compared with acidic medium (pH 1.2) which is hydrogen bonding resulted in additional cross-linking which in turn
attributed to the sulfonic anions (-SO3-) of AMPS (2-acrylamido-2- resulted in decreased swelling of nanomatrices by forming denser

10
K.U. Khan et al. Journal of Drug Delivery Science and Technology 67 (2022) 102952

Fig. 12. Comparative plasma drug concentration vs time profile of OLN and OLN-loaded nanomatrices formulation after oral administration. Each value represents
mean ± SD (n = 6).

6.6. Solubility study of nanomatrices

Table 7
Mean pharmacokinetic values of OLN and OLN-loaded nanomatrices formula­
Fig. 9 presented the solubility of drug olanzapine (OLN) in distilled
tion after oral administration to rabbits of group-B.
water, HCl solution of pH 1.2 and phosphate buffer of pH 6.8. To
Sr. No Pharmacokinetic parameter OLN (powder) β-CD nanomatrices determine the solubility of OLN, excess amount of pure drug OLN and
01 Cmax (ng/ml) 391.004 ± 5.214 671.102 ± 4.33 nanomatrices formulations were dissolved in distilled water, HCl solu­
02 Tmax (hrs) 06 1.5 ± 0.31 tion (pH 1.2), and phosphate buffer (pH 6.8) separately by using mag­
03 AUC0-t (ng.h/ml) 425.33 1576.72
netic stirrer at 200 rpm for overnight at room temperature. After this,
04 AUC0-inf (ng.h/ml) 523.98 1693.53
05 AUMC0-inf (ng.h/ml) 1038.11 3681.87 solutions were filtered and checked for absorbance at lambda max 228
06 Clast (ng/ml) 008.123 ± 0.675 042.215 ± 3.528 nm using UV-spectrophotometer. Results revealed that solubility of OLN
07 Tlast (hrs) 48 16 was significantly enhanced up-to 40 times by the prepared β-cyclodex­
08 t1/2 (hrs) 16 06 trin (β-CD) based nanomatrices which is higher than previously reported
9 Cl (L/hr) 28.43 31.35
β-cyclodextrin formulations [16,88]. Olanzapine (OLN) solubility was
Values are calculated as mean ± SD (n = 6). enhanced up to 32.99, 34.63, 36.21, 36.89, 37.11, 32.12, and 24.88
times by nanomatrices formulations BNG-1 to BNG-7 respectively in
network as shown in Fig. 8 (a). In higher pH (6.8), ionization of sulfonate distilled water. In HCl solution (1.2), OLN solubility was enhanced up to
groups resulted in strong electrostatic repulsion between sulfonic anions 33.85, 34.23, 35.11, 36.45, 37.98, 29.32, and 25.77 times, and in
(-SO3-) inside nanomatrices network which lead to increased swelling phosphate buffer (6.8), the solubility of olanzapine (OLN) was enhanced
[81] (Fig. 8b). The quick swelling behavior of nanomatrices observed in up to 34.87, 35.65, 37.15, 37.92, 40.11, 30.46, and 28.55 times
5 min at both pH conditions (1.2 and 6.8) is also attributed to the hy­ respectively by nanomatrices formulations BNG-1 to BNG-7 as shown in
drophilic nature of β-CD and monomer (AMPS). The hydrophilic effect Fig. 9. At higher pH, hydroxyl groups of β-cyclodextrin (β-CD) increases
on swelling is also shown in previous studies reported by Karadag and water solubility that’s why increased solubility was observed at pH
Erdener et al. [82,83]. Moreover, nanomatrices are nano-sizes, spongy, medium of 6.8 than distilled water and acidic medium. Due to small
and soft materials which favor rapid and enhanced swelling and drug sizes, greater surface area, and highly soft and amorphous nature,
release. nanomatrices have the ability to modify the solubility of poorly soluble
The effect of β-CD, AMPS, and MBA on nanomatrices swelling drugs. Enhanced solubility of olanzapine by nanomatrices may be due to
behavior was also evaluated. In order to evaluate swelling behavior as the porous and amorphousness of drug with in grafted nanomatrices,
function of β-CD, different concentrations of β-CD (0.1 g, 1.0 g, and 1.5 enhanced olanzapine wettability, solubilizing effect of β-CD and AMPS
g) were used in nanomatrices formulations (BNG-1, BNG-2, and BNG-3) and very large surface area due to reduce particle size.
respectively by keeping AMPS and MBA constant (Table 1). The swelling
index with increase β-CD ratio in nanomatrices formulation was 6.7. Stability and percent porosity studies of nanomatrices
increased, similar pattern was observed with increase monomer (AMPS)
concentration which may be due to pH independent behavior of AMPS Table 2 presented the porosity (%) of developed nanomatrices for­
as shown in Fig. 8 [84–86]. The swelling index was significantly mulations (BNG1-BNG7). The porosity of nanomatrices was increased
decreased by increasing MBA (cross-linking agent) concentration which with increasing concentration of β-CD (BNG-1 to BNG-3) and AMPS
was attributed to the formation of denser network in gelling structure (BNG-4 & BNG-5). This was may be due to innate porous structure of
due to over cross-linking (Fig. 8). In previous study, pH switching on-off β-CD, when its concentration was increased more interconnected
semi-IPN hydrogels were investigated and similar results of MBA were channels and voids were developed which led enhanced porosity. Sec­
reported [87]. ondly, the viscosity of reaction mixture was enhanced due to increasing

11
K.U. Khan et al.
β-CD and AMPS concentration which resisted the escape of bubbles from
mixture which in turn resulted an increased porosity. Asghar and Clara
et al. also reported similar observations of β-CD and AMPS respectively
[60,89]. The porosity of nanomatrices were decreased (BNG-6 & BNG-7)
with increasing concentration of methylene bisacrylamide (MBA) which
may be due to greater physical entanglement, decreased mesh size and
formation of dense network due to high cross-linking [90].
Table 3 presented the results of stability study. The stability studies
of OLN-loaded nanomatrices indicated that formulation remained stable
during the specified time period. No noticeable changes were observed
in physical appearance, drug loaded content (%DLC), FTIR analysis and
solubilization efficiency which confirmed the stability of nanomatrices
formulation.
6.8. In-vitro dissolution and percent drug entrapment efficiency (%DEE)
of nanomatrices
In-vitro dissolution studies were carried out at HCl solution of pH 1.2
and phosphate buffer of pH 6.8 to determine the in-vitro drug olanzapine
(OLN) release in different pH conditions. Dissolution studies were per­
formed for all prepared nanomatrices formulations (BNG-1 to BNG-7)
and for commercially available tablet (OLANZIA®) used as reference
product for OLN in order to compare drug release. A significant differ­
ence in release of OLN was observed from the reference product and
prepared nanomatrices formulations at both pH conditions as shown in
Fig. 10 (a, b). Excellent release was noticed at all pH medium but more
quickly in phosphate buffer of pH 6.8 than HCl solution of pH 1.2 i.e.,
≥90% drug (OLN) was released in just 2–10 min at higher pH (6.8) and
5–15 min at lower pH (1.2). Although OLN is a week basic drug that
favor dissolution at lower pH condition (1.2), however increase and
rapid drug release at higher pH condition (6.8) was attributed to several
factors such as (a) higher swelling index of nanomatrices at high pH
condition as drug release mechanism is directly related to swelling, (b)
protonation of sulfonate anions (-SO3-) of AMPS (2-acrylamido-2-methyl
propane sulfonic acid) into –SO3H which resulted additional cross-
linking between sulfonate groups that cause decreased swelling and
drug release at lower pH (1.2), (c) electrostatic repulsion between sul­
fonate anions (-SO3-) at higher pH resulted higher swelling as well as
drug release as it is well known that swelling of nanomatrices is induced
by electrostatic repulsion of the ionic charges in its gelling network [91,
92], (d) β-cycyldextrin (β-CD) possess hydroxyl (-OH) and hydrox­
ymethyl (CH2OH) groups which impart hydrophilicity, at low pH these
groups remained unionized due to which less swelling and release was
observed while at higher pH of 6.8 these groups were ionized resulted in
increased repulsive forces which in-turn lead to increased swelling,
water uptake and hence drug release [93,94] as shown in Fig. 10 (a, b).
Besides pH effect on dissolution, the effect of β-cylodextrin (β-CD),
monomer (AMPS), and cross-linking agent (MBA) feed contents were
also evaluated. In first three formulations BNG-1, BNG-2, and BNG-3 the
feed ratio of β-CD was increased i.e., 0.1 g, 01 g, and 1.5 g respectively
while kept the concentration of other reactants (AMPS and MBA) con­
stant as shown in Table 1. There was observed a gradual increase in drug
release with increase in β-CD concentration, similar pattern was fol­
lowed by AMPS (monomer) (BNG-4 and BNG-5) as shown in Fig. 10.
This was attributed to the water loving (hydrophilic) properties of β-CD
and AMPS that favor swelling of nanomatrices and due to the presence of
high cross-linking points in the system. In a previous study, hydrox­
ypropyl-β-CD hybrid nanomatrices were investigated and similar results
were reported [59]. In formulations BNG-6 and BNG-7, the swelling as
well as drug release was significantly decreased by increasing MBA
(cross-linking agent) concentration which was attributed to the forma­
tion of denser network in gelling structure due to over cross-linking [87]
12
Journal of Drug Delivery Science and Technology 67 (2022) 102952
as shown in Fig. 10. The percent drug loaded content (%DLC) and
entrapment efficiency (%EE) was also increased with increase feed ratio
of β-CD and AMPS (BNG1 to BNG5) and decreased with MBA feed
concentration (BNG-6 and BNG-7) as shown in Table 1. %EE up to 93%
guaranteed the efficiency of nanomatrices preparation technique.
After evaluation of pH and reactants effect on dissolution, OLN-
loaded nanomatrices formulation was also evaluated with the refer­
ence product of olanzapine (OLANZIA®) at both pH conditions of 1.2
and 6.8. Results revealed that about 90% drug was released from the
reference product in 40–60 min as compared with nanomatrices for­
mulations where more than 90% was release in just 2–10 min. The
significant difference with reference product (OLANZIA®) confirmed
that solubility, and release characteristics of OLN was distinctly
enhanced by the nanomatrices.
Nanomatrices rapid response to aqueous medium is its unique
characteristics that differentiate this specialized system from the other
reported drug delivery systems for solubility enhancement of poorly
soluble drugs. Upon contact with aqueous medium, nanomatrices
abruptly swell and the entrapped drug immediately release which pre­
sents itself as fine particles for rapid dissolution. Higher swelling, greater
drug loading capacity, higher solubility and excellent release profile
could describe the solubility enhancement mechanism of these system.
Mechanism of drug release from nanomatrices is illustrated in Fig. 10
(c).
6.9. In-vivo cytotoxicity evaluation
In order to check the safety and biocompatibility of the prepared
nanomatrices drug delivery system with the biological enivronment,
toxicity study was performed using rabbits as test animals. The study
protocols were reviewed and granted under reference number 14/2020/
PAEC by the institutional committee i.e., Pharmacy Animal Ethics
Committee (PAEC), Faculty of Pharmacy, Department of Pharmaceutics,
The Islamia University of Bahawalpur (IUB) Pakistan. The procedure for
toxicity study is mentioned in materials and method chapter. Different
physiological parameters were keenly observed during study such as;
water and food intake, salivation, tremors, urination, diarrhea, illness,
response behavior, any irritation (ocular/dermal), and mortality.
Aforementioned clinical observations are presented in Table 4 and no
perceptible change was noticed.
After 15th day blood samples of both groups (control/treated) were
collected and preserved in ethylene diamine tetra acetic acid tubes
(EDTA) for the evaluation of serum and CBC (complete blood count)
analysis. Newly polymeric system of nanomatrices may contain un-
reactant contents (β-CD/AMPS/MBA/APS) that may cause toxicity to
host cells. Animals were then sacrificed by slaughtering for histopath­
ological examination of vital organs (heart, liver, lungs, kidneys, stom­
ach, and colon). Lab results of hematological and biochemical analysis
of blood and liver function are shown in Tables 5 and 6 respectively. As
all findings were in normal ranges and no drastic change was observed
that confirmed the safety of developed beta-cyclodextrin based
nanomatrices.
In order to evaluate the effect of prepared nanomatrices on vital
organs, animals were sacrificed and histopathological micrographs at
different magnifications were captured under optical microscope. Vital
organs (heart, liver, lungs, kidneys, stomach, spleen, and colon) were
preserved in 10% formalin solution and tissue slides were made for gross
histopathological tissue examination. Microscopic examination of vital
organs depicted no signs of ulceration, inflammatory infiltration, hy­
pertrophy, lesion, and mucosal damage etc., as shown in Fig. 11.
Conclusively, the developed β-CD-co-poly (AMPS) nanomatrices was
found to be safe, and bio-compatible to the biological environment.
K.U. Khan et al.
6.10. In-vivo pharmacokinetic evaluation
6.10.1. Mean plasma olanzapine concentration time profile and its
pharmacokinetic parameters
Mean plasma concentration of OLN vs time was calculated after oral
administration of OLN powder, and OLN loaded β-cyclodextrin (β-CD)
based nanomatrices formulation as shown in Fig. 12. Overall results
revealed that there was significant difference in plasma concentration
time curve of OLN loaded β-CD based nanomatrices and OLN powder. It
was indicated from the results that there would be an obvious difference
in rate and extent of OLN absorption.
Various pharmacokinetic parameters i.e., Cmax, Tmax, AUC, Clast,
Tlast, t1/2, and Cl were calculated using PK-Solver add in program and
Microsoft Excel (2013). Results of comparative pharmacokinetic pa­
rameters are given in Table 7. Maximum plasma drug concentration
(Cmax) value was observed as 671.102 ng/ml after oral administration
of loaded β-cyclodextrin (β-CD) nanomatrices formulation which was
significantly higher as compare to the Cmax observed after oral
administration of OLN (391.004). Time to reach maximum drug con­
centration (Tmax) for (β-CD) nanomatrices formulation was noted 1.5 h
as compared to OLN powder (6 h) as presented in Table 7. Half-life (t1/2)
for β-CD formulation was calculated as 6 h while half-life for OLN given
orally was calculated as 16 h as shown in Table 7.
7. Conclusion
It is well known that various pharmaceutical industries are facing the
common issue of poor aqueous solubility because currently 40% of the
marketed and about 70–90% of new chemical entities under develop­
ment stage are poor soluble that results in reduce therapeutic effects and
dosage escalation. To address the issue, in present work, β-CD based
nanomatrices has been successfully prepared using cross-linking poly­
merization with subsequent condensatin technique. The FITR and XRD
analysis indicated the formation of cross-linked network and incorpo­
ration of drug in the amorphous system of nanomatrices which is an
important factor in solubilization enhancement. Solubility studies
revealed that the solubility of poorly soluble drug OLN was enhanced up
to 40.11 times by the prepared nanomatrices. The in-vitro dissolution
experiment demonstrated enhanced and rapid release of OLN in both pH
condition (1.2 and 6.8) as compared to the reference product (OLAN­
ZIA®). Toxicity study endorsed the safety of nanomatrices to the bio­
logical system while HPLC study revealed that pharmacokinetic profile
of OLN has been significantly improved. Conclusively, prepared β-CD
based nanomatrices carrier system is non-toxic, safe, economical, and
has no affect on body vital organs of rabbits, based on these results, the
devloped nanomatrices could be an effective strategy for enhancing
solubility, bioavailability, and therapeutic effects of poorly soluble
drugs.
Declaration of interests
The authors report no conflict of interests.
Declaration of interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This research was supported by the Higher Education Commission
(HEC) of Pakistan.
13
Journal of Drug Delivery Science and Technology 67 (2022) 102952
References
[1] A.R. Choudhury, Synthesis of a novel gellan-pullulan nanogel and its application in
adsorption of cationic dye from aqueous medium, Carbohydr. Polym. 227 (2020)
115291.
[2] S.F. Badshah, et al., Porous and highly responsive cross-linked β-cyclodextrin based
nanomatrices for improvement in drug dissolution and absorption, Life Sci. 267
(2021) 118931.
[3] Richa, A. Roy Choudhury, Synthesis of a novel gellan-pullulan nanogel and its
application in adsorption of cationic dye from aqueous medium, Carbohydr.
Polym. 227 (2020) 115291.
[4] Y. Zhang, et al., Off-Stoichiometric Thiol-Ene Chemistry to Dendritic Nanogel
Therapeutics, Advanced Functional Materials, 2019, p. 1806693.
[5] R. Kandil, O.M. Merkel, Recent progress of polymeric nanogels for gene delivery,
Curr. Opin. Colloid Interface Sci. 39 (2019) 11–23.
[6] M.A. Grimaudo, A. Concheiro, C. Alvarez-Lorenzo, Nanogels for regenerative
medicine, J. Contr. Release 313 (2019) 148–160.
[7] F. Mahmoodzadeh, M. Ghorbani, B. Jannat, Glutathione and pH-responsive
chitosan-based nanogel as an efficient nanoplatform for controlled delivery of
doxorubicin, J. Drug Deliv. Sci. Technol. 54 (2019) 101315.
[8] P. Prabhu, V. Patravale, Dissolution enhancement of atorvastatin calcium by co-
grinding technique, Drug Deliv. Translat. Res. 6 (4) (2016) 380–391.
[9] M. Sharma, et al., Enhancement of oral bioavailability of poorly water soluble
carvedilol by chitosan nanoparticles: optimization and pharmacokinetic study, Int.
J. Biol. Macromol. 135 (2019) 246–260.
[10] Y. Liu, et al., Dissolution and oral bioavailability enhancement of praziquantel by
solid dispersions, Drug Deliv. Translat. Res. 8 (3) (2018) 580–590.
[11] B. Dong, K. Hadinoto, Carboxymethyl cellulose is a superior polyanion to dextran
sulfate in stabilizing and enhancing the solubility of amorphous drug-
polyelectrolyte nanoparticle complex, Int. J. Biol. Macromol. 139 (2019) 500–508.
[12] M. Imono, et al., The elucidation of key factors for oral absorption enhancement of
nanocrystal formulations: in vitro–in vivo correlation of nanocrystals, Eur. J.
Pharm. Biopharm. 146 (2020) 84–92.
[13] A. Naqvi, et al., Preparation and evaluation of pharmaceutical co-crystals for
solubility enhancement of atorvastatin calcium, Polym. Bull. (2019).
[14] D. Morina, et al., Oral tablet formulations containing cyclodextrin complexes of
poorly water soluble cefdinir to enhance its bioavailability, J. Drug Deliv. Sci.
Technol. 57 (2020) 101742.
[15] K.U. Khan, et al., Synthesis of PEG-4000-Co-Poly (AMPS) Nanogels by Cross-
Linking Polymerization as Highly Responsive Networks for Enhancement in
Meloxicam Solubility, Drug Development and Industrial Pharmacy, 2021,
pp. 1–12.
[16] Q. Khalid, et al., Novel β-cyclodextrin nanosponges by chain growth condensation
for solubility enhancement of dexibuprofen: characterization and acute oral
toxicity studies, J. Drug Deliv. Sci. Technol. (2020) 102089.
[17] Q. Fu, et al., Salt formation of two BCS II drugs (indomethacin and naproxen) with
(1R, 2R)-1, 2-diphenylethylenediamine: crystal structures, solubility and
thermodynamics analysis, J. Mol. Struct. 1185 (2019) 281–289.
[18] J. Xu, et al., Azilsartan piperazine salt solvate and monohydrate: preparation,
crystal structure, enhanced solubility and oral bioavailability, New J. Chem.
(2020).
[19] X. Shi, et al., Quercetin Amorphous Solid Dispersions Prepared by Hot Melt
Extrusion with Enhanced Solubility and Intestinal Absorption, Pharmaceutical
Development and Technology, 2020, pp. 1–10.
[20] A. Tambe, N. Pandita, Enhanced solubility and drug release profile of boswellic
acid using a poloxamer-based solid dispersion technique, J. Drug Deliv. Sci.
Technol. 44 (2018) 172–180.
[21] Y. Chen, et al., Fabrication and characterization of zein/lactoferrin composite
nanoparticles for encapsulating 7, 8-dihydroxyflavone: enhancement of stability,
water solubility and bioaccessibility, Int. J. Biol. Macromol. 146 (2020) 179–192.
[22] B. Niu, et al., Hydrophobin-enhanced stability, dispersions and release of curcumin
nanoparticles in water, J. Biomater. Sci. (2020) 1–16 (just-accepted), Polymer
Edition.
[23] R. Diwan, et al., Cilnidipine Loaded Poly (ε-Caprolactone) Nanoparticles for
Enhanced Oral Delivery: Optimization Using DoE, Physical Characterization,
Pharmacokinetic and Pharmacodynamic Evaluation, Pharmaceutical development
and technology, 2021, pp. 1–43.
[24] Y. Ikeuchi-Takahashi, et al., Development of microparticles coated with poly-
γ-glutamic acid to improve oral absorption of a poorly water-soluble drug,
Pharmaceut. Dev. Technol. 24 (8) (2019) 992–1001.
[25] Y. Bi, et al., A liposomal formulation for improving solubility and oral
bioavailability of nifedipine, Molecules 25 (2) (2020) 338.
[26] A. Alshweiat, et al., Nasal delivery of nanosuspension-based mucoadhesive
formulation with improved bioavailability of loratadine: preparation,
characterization, and in vivo evaluation, Int. J. Pharm. (2020) 119166.
[27] M. Gao, et al., Enhanced Curcumin Solubility and Antibacterial Activity by
Encapsulation in PLGA Oily Core Nanocapsules, Food Funct. (2020).
[28] C. Sun, et al., Development of TPGS/F127/F68 mixed polymeric micelles:
enhanced oral bioavailability and hepatoprotection of syringic acid against carbon
tetrachloride-induced hepatotoxicity, Food Chem. Toxicol. (2020) 111126.
[29] J. Liang, et al., Enhanced solubility and targeted delivery of curcumin by
lipopeptide micelles, J. Biomater. Sci. Polym. Ed. 26 (6) (2015) 369–383.
[30] J. Mao, et al., Transdermal delivery of rapamycin with poor water-solubility by
dissolving polymeric microneedles for anti-angiogenesis, J. Mater. Chem. B (2020).
[31] F. Najafi, et al., A comparative study on solubility improvement of tetracycline and
dexamethasone by poly (propylene imine) and polyamidoamine dendrimers: an
K.U. Khan et al.
insight into cytotoxicity and cell proliferation, J. Biomed. Mater. Res. 108 (3)
(2020) 485–495.
[32] S. Potharaju, et al., Improving Solubility and Oral Bioavailability of a Novel
Antimalarial Prodrug: Comparing Spray-Dried Dispersions with Self-Emulsifying
Drug Delivery Systems, Pharmaceutical Development and Technology, 2020,
pp. 1–47 (just-accepted).
[33] J. Ke, et al., Novel chiral composite membrane prepared via the interfacial
polymerization of diethylamino-beta-cyclodextrin for the enantioseparation of
chiral drugs, J. Membr. Sci. 597 (2020) 117635.
[34] M. Salih, et al., Supramolecular amphiphiles of Beta-cyclodextrin and Oleylamine
for enhancement of vancomycin delivery, Int. J. Pharm. 574 (2020) 118881.
[35] B.S. Verza, et al., A long-term controlled drug-delivery with anionic beta
cyclodextrin complex in layer-by-layer coating for percutaneous implants devices,
Carbohydr. Polym. 257 (2021) 117604.
[36] U. Gupta, et al., Dendrimers: novel polymeric nanoarchitectures for solubility
enhancement, Biomacromolecules 7 (3) (2006) 649–658.
[37] B. Gidwani, A. Vyas, Pharmacokinetic study of solid-lipid-nanoparticles of
altretamine complexed epichlorohydrin-β-cyclodextrin for enhanced solubility and
oral bioavailability, Int. J. Biol. Macromol. 101 (2017) 24–31.
[38] D. Sid, et al., Solubility enhancement of mefenamic acid by inclusion complex with
β-cyclodextrin: in silico modelling, formulation, characterisation, and in vitro
studies 36 (1) (2021) 605–617.
[39] T. Loftsson, et al., Cyclodextrins in drug delivery, Expet Opin. Drug Deliv. 2 (2)
(2005) 335–351.
[40] D.-H. Kim, et al., Solubility enhancement and application of cyclodextrins in local
drug delivery, J. Pharmaceut. Investig. 50 (1) (2020) 17–27.
[41] A. Martini, et al., Use of dehydrated beta-cyclodextrin as pharmaceutical excipient,
Drug Dev. Ind. Pharm. 20 (15) (1994) 2381–2393.
[42] B. Gidwani, A. Vyas, A comprehensive review on cyclodextrin-based carriers for
delivery of chemotherapeutic cytotoxic anticancer drugs, BioMed Res. Int. 2015
(2015), 198268-198268.
[43] J.N. BeMiller, R.L. Whistler, Starch: Chemistry and Technology, Academic Press,
2009.
[44] T. Zhuang, et al., Isolation, identification and characterization of two novel
process-related impurities in olanzapine, J. Pharmaceut. Biomed. Anal. 152 (2018)
188–196.
[45] S.R.S. Rudrangi, et al., Preparation of olanzapine and methyl-β-cyclodextrin
complexes using a single-step, organic solvent-free supercritical fluid process: an
approach to enhance the solubility and dissolution properties, Int. J. Pharmaceut.
494 (1) (2015) 408–416.
[46] K.N. Günther, et al., Segmental hair analysis of olanzapine and N-desmethyl-
olanzapine in postmortem hair from mentally ill patients by LC–MS/MS,
J. Pharmaceut. Biomed. Anal. 190 (2020) 113510.
[47] N. Jawahar, et al., Enhanced oral bioavailability of an antipsychotic drug through
nanostructured lipid carriers, Int. J. Biol. Macromol. 110 (2018) 269–275.
[48] J.G. Pontes-Neto, et al., Evaluation of antioxidant potential of novel CaAl and NiAl
layered double hydroxides loaded with olanzapine, Life Sci. 207 (2018) 246–252.
[49] N.F. da Costa, A.I. Fernandes, J.F. Pinto, Measurement of the amorphous fraction of
olanzapine incorporated in a co-amorphous formulation, Int. J. Pharm. 588 (2020)
119716.
[50] E. Dziurkowska, et al., Development and validation of solid-phase extraction
coupled with a liquid chromatography-tandem mass spectrometry method for
quantitation of olanzapine in saliva, J. Chromatogr. B 1136 (2020) 121896.
[51] J.G. Pontes-Neto, et al., Intercalation of olanzapine into CaAl and NiAl Layered
Double Hydroxides for dissolution rate improvement: synthesis, characterization
and in vitro toxicity, J. Drug Deliv. Sci. Technol. 52 (2019) 986–996.
[52] N. Anup, S. Thakkar, M. Misra, Formulation of olanzapine nanosuspension based
orally disintegrating tablets (ODT); comparative evaluation of lyophilization and
electrospraying process as solidification techniques, Adv. Powder Technol. 29 (8)
(2018) 1913–1924.
[53] D. Riman, et al., The use of micro carbon pencil lead electrode for sensitive HPLC-
ED analysis of selected antipsychotic drugs, Microchem. J. 154 (2020) 104606.
[54] M.R. de Freitas, et al., Inclusion complex of methyl-β-cyclodextrin and olanzapine
as potential drug delivery system for schizophrenia, Carbohydr. Polym. 89 (4)
(2012) 1095–1100.
[55] L. Ren, et al., Chronic treatment with the modified Longdan Xiegan Tang
attenuates olanzapine-induced fatty liver in rats by regulating hepatic de novo
lipogenesis and fatty acid beta-oxidation-associated gene expression mediated by
SREBP-1c, PPAR-alpha AMPK-alpha 232 (2019) 176–187.
[56] J. Chen, et al., FADS1 modulates metabolic syndrome in schizophrenia patients
receiving olanzapine monotherapy, Asian J. Psychiatr. 54 (2020) 102352.
[57] N. Yang, et al., Identification and characterization of proteins that are differentially
expressed in adipose tissue of olanzapine-induced insulin resistance rat by iTRAQ
quantitative proteomics, J. Proteomics 212 (2020) 103570.
[58] D. Ng-Mak, et al., Hospitalization risk in bipolar disorder patients treated with
lurasidone versus other atypical antipsychotics 35 (2) (2019) 211–219.
[59] Q. Khalid, M. Ahmad, M. Usman Minhas, Hydroxypropyl-β-cyclodextrin hybrid
nanogels as nano-drug delivery carriers to enhance the solubility of dexibuprofen:
characterization, in vitro release, and acute oral toxicity studies, Adv. Polym.
Technol. 37 (6) (2018) 2171–2185.
14
Journal of Drug Delivery Science and Technology 67 (2022) 102952
[60] S. Asghar, et al., Bi-polymeric spongy matrices through cross-linking
polymerization: synthesized and evaluated for solubility enhancement of acyclovir
22 (5) (2021) 1–16.
[61] A. Naqvi, et al., Preparation and evaluation of pharmaceutical co-crystals for
solubility enhancement of atorvastatin calcium, Polym. Bull. 77 (12) (2020)
6191–6211.
[62] M. Abbasi, et al., Novel biodegradable pH-sensitive hydrogels: an efficient
controlled release system to manage ulcerative colitis, Int. J. Biol. Macromol.
(2019).
[63] World Health Organization, Stability testing of active pharmaceutical ingredients
and finished pharmaceutical products, WHO Technical report series, 2009,
pp. 87–123, 953.
[64] K.-R. Lee, et al., Effect of poloxamer on the dissolution of felodipine and
preparation of controlled release matrix tablets containing felodipine, Arch Pharm.
Res. (Seoul) 31 (8) (2008) 1023–1028.
[65] V. Mechtcherine, et al., Testing superabsorbent polymer (SAP) sorption properties
prior to implementation in concrete: results of a RILEM Round-Robin Test, Mater.
Struct. 51 (1) (2018) 28.
[66] S.A. Shah, et al., pH-responsive CAP-co-poly (methacrylic acid)-based hydrogel as
an efficient platform for controlled gastrointestinal delivery: fabrication,
characterization, in vitro and in vivo toxicity evaluation, Drug Deliv. Translat. Res.
9 (2) (2019) 555–577.
[67] S. Khan, et al., Lipid poly (ϵ-caprolactone) hybrid nanoparticles of 5-fluorouracil
for sustained release and enhanced anticancer efficacy, Life Sci. 284 (2021)
119909.
[68] H.-W. Cho, et al., Orodispersible polymer films with the poorly water-soluble drug,
olanzapine: hot-melt pneumatic extrusion for single-process 3D printing,
Pharmaceutics 12 (8) (2020) 692.
[69] S.M. Abuzar, et al., Enhancing the solubility and bioavailability of poorly water-
soluble drugs using supercritical antisolvent (SAS) process, Int. J. Pharmaceut. 538
(1–2) (2018) 1–13.
[70] S.A. Khan, et al., β-Cyclodextrin-based (IA-co-AMPS) Semi-IPNs as smart
biomaterials for oral delivery of hydrophilic drugs: synthesis, characterization, in-
Vitro and in-Vivo evaluation, J. Drug Deliv. Sci. Technol. 60 (2020) 101970.
[71] C.M. Long, et al., Surface dissolution UV imaging for investigation of dissolution of
poorly soluble drugs and their amorphous formulation, AAPS PharmSciTech 20 (3)
(2019) 113.
[72] B.C. Hancock, M. Parks, What is the true solubility advantage for amorphous
pharmaceuticals? Pharmaceut. Res. 17 (4) (2000) 397–404.
[73] N.S. Malik, M. Ahmad, M.U.J.P.o. Minhas, Cross-linked β-cyclodextrin and
carboxymethyl cellulose hydrogels for controlled drug delivery of acyclovir 12 (2)
(2017) e0172727.
[74] C. Zhang, A.J. Easteal, Study of free-radical copolymerization of N-
isopropylacrylamide with 2-acrylamido-2-methyl-1-propanesulphonic acid 88 (11)
(2003) 2563–2569.
[75] M. Ali, et al., Novel composite pH controlled drug release hydrogel containing
dexibuprofen, RADS J. Pharm. Pharmaceut. Sci. 6 (4) (2018) 223–235.
[76] L. Ali, et al., Controlled release of highly water-soluble antidepressant from hybrid
copolymer poly vinyl alcohol hydrogels, Polym. Bull. 71 (1) (2014) 31–46.
[77] L. Yin, et al., Superporous hydrogels containing poly (acrylic acid-co-acrylamide)/
O-carboxymethyl chitosan interpenetrating polymer networks, Biomaterials 28 (6)
(2007) 1258–1266.
[78] K. Ullah, et al., Gelatin-based hydrogels as potential biomaterials for colonic
delivery of oxaliplatin, Int. J. Pharmaceut. 556 (2019) 236–245.
[79] T. Fekete, et al., Synthesis of cellulose-based superabsorbent hydrogels by high-
energy irradiation in the presence of crosslinking agent, Radiat. Phys. Chem. 118
(2016) 114–119.
[80] N.M. Ranjha, et al., Preparation and characterization of hybrid pH-sensitive
hydrogels of chitosan-co-acrylic acid for controlled release of verapamil, J. Mater.
Sci. Mater. Med. 21 (10) (2010) 2805–2816.
[81] E. Karadağ, S. Kundakci, Ö. Barış Üzüm, Water sorption and dye uptake studies of
highly swollen AAm/AMPS hydrogels and semi-IPNs with PEG, Polym. Plast.
Technol. Eng. 48 (12) (2009) 1217–1229.
[82] E. Karadağ, et al., Uranyl ion sorption characteristics of novel polymer/
montmorillonite/carboxymethyl cellulose-composite biosorbent-based AA m/
AMPS hydrogels and semi-IPN s, Adv. Polym. Technol. 37 (2) (2018) 575–585.
[83] E. Karadağ, et al., Swelling equilibria of novel propenamide/2-acrylamido-2-
methyl-1-propanesulfonic acid/guar gum/clinoptilolite biohybrid hydrogels and
application as a sorbent for BV1 removal, Polym. Bull. (2020).
[84] M.U. Minhas, et al., Functionalized pectin hydrogels by cross-linking with
monomer: synthesis, characterization, drug release and pectinase degradation
studies, Polym. Bull. (2019) 1–18.
[85] Ö.B. Üzüm, et al., Swelling behaviors of novel magnetic semi-IPN hydrogels and
their application for Janus Green B removal, Polym. Bull. (2019) 1–21.
[86] M.F. Broglia, et al., Acid Hydrogel Matrixes as Reducing/stabilizing Agent for the
In-Situ Synthesis of Ag-Nanocomposites by UV Irradiation: pH Effect, Materials
Research Express, 2019.
[87] Y. Zhang, et al., pH switching on-off semi-IPN hydrogel based on cross-linked poly
(acrylamide-co-acrylic acid) and linear polyallyamine, Polymer 46 (18) (2005)
7695–7700.
K.U. Khan et al.
[88] S.F. Badshah, et al., Porous and Highly Responsive Cross-Linked β-cyclodextrin
Based Nanomatrices for Improvement in Drug Dissolution and Absorption, Life Sci
(2020) 118931.
[89] I. Clara, R. Lavanya, N.J. Natchimuthu, Part A, pH and temperature responsive
hydrogels of poly (2-acrylamido-2-methyl-1-propanesulfonic acid-co-methacrylic
acid), Synth. Swell. Charact. 53 (8) (2016) 492–499.
[90] K. Sohail, et al., pH-sensitive polyvinylpyrrolidone-acrylic acid hydrogels, Impact
Mater. Parameters Swell. Drug Release 50 (2014) 173–184.
[91] M.A. Taleb, D.E. Hegazy, G.A. Mahmoud, Characterization and in vitro drug
release behavior of (2-hydroxyethyl methacrylate)–co-(2-acrylamido-2-methyl-1-
15
Journal of Drug Delivery Science and Technology 67 (2022) 102952
propanesulfonic acid) crosslinked hydrogels prepared by ionizing radiation, Int. J.
Polym. Mater. Polym. Biomater. 63 (16) (2014) 840–845.
[92] C. Bode, et al., Often Neglected: PLGA/PLA Swelling Orchestrates Drug Release:
HME Implants, J. Control. Release (2019).
[93] N.S. Malik, M. Ahmad, M.U. Minhas, Cross-linked β-cyclodextrin and
carboxymethyl cellulose hydrogels for controlled drug delivery of acyclovir, PLoS
One 12 (2) (2017) e0172727.
[94] S. Swaminathan, et al., Nanosponges encapsulating dexamethasone for ocular
delivery: formulation design, physicochemical characterization, safety and corneal
permeability assessment, J. Biomed. Nanotechnol. 9 (6) (2013) 998–1007.