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A novel pH‐responsive nanoniosomal emulsion for sustained release of curcumin from a chitosan‐based nanocarrier: Emphasis on the concurrent improvement of loading, sustained release, and apoptosis induction

Biotechnology Progress

June 2022
38(5)

DOI:10.1002/btpr.3280

Authors:

Mehrab Pourmadadi

Shahid Beheshti University

Amirmasoud Samadi

University of California, Irvine

Show all 7 authorsHide

Curcumin application as an anti‐cancer drug is faced with several impediments. This study has developed a platform that facilitates the sustained release of curcumin, improves loading efficiency, and anti‐cancer activity. Montmorillonite (MMT) nanoparticles were added to chitosan (CS)‐agarose (Aga) hydrogel and then loaded with curcumin (Cur) to prepare a curcumin‐loaded nanocomposite hydrogel. The loading capacity increased from 63% to 76% by adding MMT nanoparticles to a chitosan‐agarose hydrogel. Loading the fabricated nanocomposite in the nanoniosomal emulsion resulted in sustained release of curcumin under acidic conditions. Release kinetics analysis showed diffusion and erosion are the dominant release mechanisms, indicating non‐fickian (or anomalous) transport based on the Korsmeyer‐Peppas model. FTIR spectra confirmed that all nanocomposite components were present in the fabricated nanocomposite. Besides, XRD results corroborated the amorphous structure of the prepared nanocomposite. Zeta potential results corroborated the stability of the fabricated nanocarrier. Cytotoxicity of the prepared CS‐Aga‐MMT‐Cur on MCF‐7 cells was comparable with that of curcumin‐treated cells (p < 0.001). Moreover, the percentage of apoptotic cells increased due to the enhanced release profile resulting from the addition of MMT to the hydrogel and the incorporation of the fabricated nanocomposite into the nanoniosomal emulsion. To recapitulate, the current delivery platform improved loading, sustained release, and curcumin anti‐cancer effect. Hence, this platform could be a potential candidate to mitigate cancer therapy restrictions with curcumin.

Schematics of crosslinking between (a) chitosan and chitosan, (b) chitosan and agarose, and (c) agarose and agarose using glyoxal as a crosslinker

…

FTIR spectra of the samples

…

XRD patterns of the samples

…

FESEM of lyophilized nanocomposite hydrogels of CS‐Aga‐MMT loaded with curcumin

…

Size distribution of CS‐Aga‐MMT‐Cur nanocarrier

…

Figures - available from: Biotechnology Progress

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RESEARCH ARTICLE

Formulation and Engineering of Biomaterials

A novel pH-responsive nanoniosomal emulsion for sustained

release of curcumin from a chitosan-based nanocarrier:

Emphasis on the concurrent improvement of loading, sustained

release, and apoptosis induction

Shabnam Haseli

| Mehrab Pourmadadi

| Amirmasoud Samadi

Fatemeh Yazdian

| Majid Abdouss

| Hamid Rashedi

| Mona Navaei-Nigjeh

4,5

Department of Biotechnology, School of

Chemical Engineering, College of Engineering,

University of Tehran, Tehran, Iran

Department of Life Science Engineering,

Faculty of New Science and Technologies,

University of Tehran, Tehran, Iran

Department of Chemistry, Amirkabir

University of Technology, Tehran, Iran

Pharmaceutical Sciences Research Center,

The Institute of Pharmaceutical Sciences

(TIPS), Tehran University of Medical Sciences,

Tehran, Iran

Department of Pharmaceutical Biomaterials

and Medical Biomaterials Research Center,

Faculty of Pharmacy, Tehran University of

Medical Sciences (TUMS), Tehran, Iran

Correspondence

Fatemeh Yazdian, Department of Life Science

Engineering, Faculty of New Science and

Technologies, University of Tehran, Tehran,

Iran.

Email: yazdian@ut.ac.ir

Hamid Rashedi, Department of Biotechnology,

School of Chemical Engineering, College of

Engineering, University of Tehran, Tehran,

Iran.

Email: hrashedi@ut.ac.ir

Abstract

Curcumin application as an anti-cancer drug is faced with several impediments. This

study has developed a platform that facilitates the sustained release of curcumin,

improves loading efficiency, and anti-cancer activity. Montmorillonite (MMT) nano-

particles were added to chitosan (CS)-agarose (Aga) hydrogel and then loaded with

curcumin (Cur) to prepare a curcumin-loaded nanocomposite hydrogel. The loading

capacity increased from 63% to 76% by adding MMT nanoparticles to a chitosan-

agarose hydrogel. Loading the fabricated nanocomposite in the nanoniosomal emul-

sion resulted in sustained release of curcumin under acidic conditions. Release kinet-

ics analysis showed diffusion and erosion are the dominant release mechanisms,

indicating non-fickian (or anomalous) transport based on the Korsmeyer-Peppas

model. FTIR spectra confirmed that all nanocomposite components were present in

the fabricated nanocomposite. Besides, XRD results corroborated the amorphous

structure of the prepared nanocomposite. Zeta potential results corroborated the

stability of the fabricated nanocarrier. Cytotoxicity of the prepared CS-Aga-MMT-

Cur on MCF-7 cells was comparable with that of curcumin-treated cells (p< 0.001).

Moreover, the percentage of apoptotic cells increased due to the enhanced release

profile resulting from the addition of MMT to the hydrogel and the incorporation of

the fabricated nanocomposite into the nanoniosomal emulsion. To recapitulate, the

current delivery platform improved loading, sustained release, and curcumin anti-

cancer effect. Hence, this platform could be a potential candidate to mitigate cancer

therapy restrictions with curcumin.

KEYWORDS

cancer therapy, chitosan, curcumin, montmorillonite, niosome, pH-sensitive nanocarrier

1|INTRODUCTION

During the last decade, cancer has skyrocketed alarmingly fast as the

world's second cause of fatality across the globe.

It is estimated that

there are 1.8 million new diagnosed cases of cancer, with over

600 thousand deaths in the United States alone.

2,3

It is approximated

that breast cancer makes up 30% of all cancers diagnosed in the

United States; the most prevalent cancer among women after skin

Received: 11 April 2022 Revised: 30 April 2022 Accepted: 11 May 2022

DOI: 10.1002/btpr.3280

https://doi.org/10.1002/btpr.3280

15206033, 0, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btpr.3280 by University Of California - Irvine, Wiley Online Library on [12/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

cancer.

Multiple cancer treatments, including chemotherapy, have

been tried; however, several impediments, including toxicity to

healthy cells and cardiac tissues, multiple drug resistance (MDR),

non-specific distribution, and destructive damages on normal tissues

hamper the effective treatment of cancer with chemotherapy.

5,6

Therefore, safer anti-cancer drugs, and efficient, targeted delivery

methods are needed. In this direction, taking advantage of the anti-

cancer properties of naturally derived polyphenols such as curcumin

and delivering it via stimuli-responsive delivery systems has opened

up a new avenue for cancer therapy.

7,8

Curcumin (Cur), a plant-derived flavonoid polyphenol, is one of the

main constituents of food and beverages.

Curcumin has several fea-

tures, involving anti-inflammatory,

antiviral,

anti-Leishmanial

activity,

and anti-cancer properties through controlling tumor progres-

sion by increasing reactive oxygen species (ROS) after targeting ROS

metabolic pathway enzymes.

Several physiological activities have been

ascribed to curcumin for controlling tumor progression, including inhibi-

tion of the WNT/β-catenin pathway and pyruvate kinase M2

(PKM2).

13,14

Curcumin is also utilized as a chemosensitizer to reduce

cancer cells' multidrug resistance by blocking the nuclear factor NF-κB.

The mentioned attributes render curcumin a promising compound

for cancer treatment; however, low solubility(1μg.ml

1

), instability,

short biological half-life, and poor permeability are among the striking

negative aspects that impede employing anti-cancer properties of curcu-

min.

16,17

The aforementioned deficiencies of curcumin for cancer therapy

can be mitigated with the fabrication of hydrogels using natural polymers

as a promising approach for employing hydrogels in drug delivery applica-

tions.

18,19

In this study, the hydrogel is prepared by chitosan and agarose.

Chitosan (CS) is a biocompatible biopolymer comprised of

β-linked D-glucosamine and N-acetyl-D-glucosamine. Enhancing pro-

longed release and drug penetration, low toxicity, and structural vari-

ability are some of the properties that render chitosan a desirable

natural polymer for hydrogel fabrication in biomedical applications,

including drug delivery.

20–22

As well, chitosan amine groups proton-

ation at the low pH of cancer cells leads to the pH-sensitive release of

loaded drugs in hydrogel; on the other hand, chitosan retains its gel-

like structure at pH 7.4. Indeed, this pH-sensitive property of chitosan

renders the fabricated hydrogel pH-responsive and prevents the

release of drugs under neutral conditions before reaching tumor

cells.

For instance, Díaz-Zepeda et al. coated microemulsions con-

taining curcumin in the oil phase with chitosan to obtain a pH-

sensitive platform for curcumin delivery.

Also, due to chitosan's

semi-crystalline structure and high hydrophilicity, it forms hydrogen

bonds with other compounds within the hydrogel network, includ-

ing agarose. Agarose (Aga) is a porous polysaccharide with d-galac-

tose-3,6-anhydro-l-galactopyranose as building blocks.

Contrary

to PEG and alginate, which are prone to reactive chemistry and lack

of effectuation capability, agarose physical crosslinking presents an

attractive property for hydrogel preparation.

Agarose-based bio-

materials, e.g., hydrogels, can be a matrix for the incorporation of

pH-sensitive compounds like diblock copolymers and chitosan to

produce pH-sensitive delivery platforms.

21,27

Indeed, agarose has

been employed as a matrix to produce pH-sensitive carriers.

28,29

Consequently, agarose is suitable for fabricating controlled stimuli-

responsive drug delivery systems.

The lack of multiresponsiveness and low loading efficiency of cur-

cumin due to hydrogels' hydrophilic nature are among the major factors

limiting their use in curcumin delivery as a hydrophobic drug. To

address these shortcomings, nanoparticles are incorporated into hydro-

gels to prepare nanocomposite hydrogels.

30,31

Nanoparticles' unique

properties, including high surface area, controlled shape, reduced parti-

cle size, and the potential to be loaded with hydrophobic molecules,

render their incorporation into hydrogels an apt solution to extend the

capacity of hydrogels for the delivery of a broader range of therapeu-

tics, including hydrophobic anti-cancer medicines.

32,33

In this vein,

montmorillonite (MMT), a common clay mineral, which exhibits biocom-

patibility, biodegradability, and good mechanical properties,

was

incorporated into the prepared CS-Aga hydrogel. As stated for nano-

particles, MMT incorporation as a biocompatible compound in the

CS-Aga hydrogel can enhance the loading capacity of hydrogel, homo-

geneity of the hydrogel, and burst release control.

35,36

This potential of

MMT is mainly ascribed to the presence of siloxane (Si–O–Si), which

plays a pivotal role in interaction with polymers in the hydrogel struc-

ture and drugs loaded in hydrogels.

Hydroxyl groups in agarose and

curcumin can create hydrogen bonds with the oxygen of the Si–O–Si

functional group of MMT, causing a high affinity for curcumin.

38,39

These interactions between components of the nanocomposite hydro-

gel maintain nanocomposite integrity under neutral conditions.

Anti-cancer drug delivery platforms that respond to pH are

among the most popular stimuli-responsive drug delivery options.

40,41

The change in the pH of cancer cells during their proliferation is one

of the main reasons for this popularity.

42,43

This reduction in pH,

known as the Warburg effect, is a hallmark in cancer and has led to

applying pH-sensitive drug delivery platforms like hydrogels for the

controlled delivery of anti-cancer therapeutics.

44,45

As mentioned before, a hydrogel of CS-Aga was prepared in this

study. MMT nanoparticles were embedded in this hydrogel to pro-

duce a CS-Aga-MMT nanocomposite hydrogel. Hydroxyl groups in

agarose provided gelling property in the hydrogel, known as physical

crosslinking property. Besides, the H-atoms of the C–H group of chit-

osan and its amine groups, agarose and curcumin hydroxyl groups,

and the oxygen atoms of the siloxane group of MMT can form hydro-

gen bonds.

Indeed, MMT nanoparticles were non-covalently incor-

porated into the hydrogel before the addition of a crosslinking agent.

Then, curcumin was added to the nanocomposite hydrogel, which led

to hydrogen bonding between MMT and curcumin.

Afterwards,

glyoxal was used as a crosslinker to further entrap the drug inside the

CS-Aga-MMT nanocomposite hydrogel by forming covalent bonds in

the hydrogel 3D network. The prepared hybrid hydrogel includes

chitosan-chitosan, chitosan-agarose, and agarose-agarose crosslinks

using glyoxal. Due to these interactions and the high surface area pro-

vided by MMT nanoparticles, curcumin loading is improved as a

hydrophobic compound in the fabricated nanocomposite hydrogel.

Furthermore, the fabricated nanocomposite has pH-responsive prop-

erty due to the alterations in the structure of nanocomposite com-

pounds with the change in pH. Indeed, Al and Si of MMT dissolve

2of18 HASELI ET AL.

faster under acidic conditions as the pH decreases.

Also, amine

groups in chitosan, hydroxyl groups in agarose and curcumin are pro-

tonated in acidic media. The release of Al and Si ions along with

protonation in the mentioned groups provides repulsive forces

between nanocomposite components. These repulsive forces lead to

the disintegration of the nanocomposite and faster release in an acidic

environment; however, the interactions between the drug and

components preserve the nanocomposite structure under neutral con-

ditions. The nanocomposite can release the drug at the tumor location

with a reduced pH by retaining the drug under neutral conditions.

Since the controlled release of curcumin and prolonging the release

time to enhance the apoptosis induction of curcumin was a major

objective of this study, the curcumin-loaded nanocomposite hydrogel

was then encapsulated in a nanoniosomal emulsion. The nanoniosomal

emulsion is a bilayer vesicle produced by a non-ionic surfactant and an

oil phase as per the steps in the literature.

47,48

The prolonged release

by encapsulating the nanocomposite in the niosome is due to separat-

ing the aqueous phase with an oil phase. Indeed, the prolonged release

is achieved since the oil layer separates the internal and external aque-

ous phases. The oil layer functions like a liquid membrane, reducing cur-

cumin release from the internal aqueous phase.

Comparing the

current study with our previous research reveals that loading the drug-

loaded nanocomposite in the nanoniosome prolongs the release.

Sev-

eral attributes make niosomes suitable platforms for curcumin delivery,

including high stability and cheaper cost of production.

The nanoniosomal emulsion is based on a water in oil emulsion

that contains the drug-loaded nanocomposite in its aqueous phase.

Span 80 is used as a non-ionic surfactant to form the first layer of the

vesicle. Then, the formed vesicle is added to the water phase to form

niosomes as double-layer vesicles. The water phase also washes the

free unbound drug from niosomes.

Hence, by incorporating MMT nanoparticles in the pH-responsive

CS-Aga hydrogel, curcumin loading was improved. Then, CS-Aga-MMT

nanocomposite hydrogel containing curcumin was encapsulated in a

niosomal emulsion to enhance the prolonged release of curcumin from

the nanocomposite. We inspected the in vitro antitumor activity of the

fabricated drug delivery system on the MCF-7 cells as a way to examine

the potential of this platform for cancer treatment. As far as we know,

this is an original method to ameliorate predominant restrictions regard-

ing cancer therapy with curcumin. This approach has the potential to

concurrently improve the loading capacity and stimuli-responsive

release at the target site within a prolonged period. Moreover, the study

of curcumin delivery by this platform demonstrated enhanced apoptosis

induction of curcumin. These results represent that the common draw-

backs of using curcumin as an anti-cancer drug, including poor loading

and low solubility, have been addressed to a good extent; thus, the use

of this platform may expand to treat other cancer cells as well.

2|MATERIALS

MCF-7 breast cancer cells were taken from the American Type Cul-

ture Collection (ATCC) (Manassas, VA). Culture flasks and dishes were

gained from Corning (Corning, NY, USA). Fetal bovine serum (FBS)

was purchased from Gibco-Life Technologies. Dulbecco's Modified

Eagle's Medium (DMEM) High Glucose was obtained from Thermo

Fisher Scientific (US). Penicillin/streptomycin and 0.25% (w/v)

trypsin–0.1% (w/v) ethylenediaminetetraacetic acid (EDTA) were pro-

cured from Thermo Fisher Scientific (US). 3-(4, 5-Dimethyl-thiazol-

2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT), and dimethylsulfoxide

(DMSO) were bought from Merck. Phosphate buffer saline (PBS) was

purchased from Sigma-Aldrich. Chitosan (CS), agarose (Aga), montmo-

rillonite (MMT), and curcumin (Cur) ((1E,6E)-1,7-bis(4-hydroxy-

3-methoxyphenyl)-1,6-heptadiene-3,5-dione) were obtained from

Sigma. Span 80 and olive oil were purchased from Sigma-Aldrich.

Glyoxal was bought from Merck.

3|METHODS

3.1 |Synthesis of CS-Aga-MMT hydrogel

At first, 30 ml of acetic acid 2% v/v was prepared to slowly add chito-

san (CS) to it at 25C with magnetic stirring (500 rpm) and prepare a

2% w/v solution. Then, agarose (1% w/v) was mixed with the former

2% w/v solution and intensely stirred. After the preparation of the

CS-Aga hydrogel, montmorillonite (MMT) was added under stirring

with 0.1% w/v. The presence of MMT in the CS-Aga hydrogel matrix

leads to physical crosslinking and the establishment of hydrogen

bonds in the nanocomposite hydrogel. These interactions result in the

enhancement of curcumin loading in the nanocomposite.

32,38

3.2 |Curcumin loading in the fabricated hydrogel

Firstly, curcumin was added to ethanol 96% v/v to dissolve it since it is

a hydrophobic compound. Then, the dissolved curcumin was incorpo-

rated into the CS-Aga-MMT nanocomposite. The model drug, curcu-

min, was added to the hydrogel with 10 μg.ml

1

concentration. The

addition of curcumin was done gradually while intensely stirred. The

nanocomposite was crosslinked once curcumin was added using glyoxal

(0.1% v/v). Crosslinking ensues from chitosan amine groups binding

together in addition to interaction with agarose hydroxyl groups. Also,

hydroxyl functional groups in agarose cause crosslinking between Aga

and Aga using glyoxal. The schematics of crosslinks between polymers

are depicted in Figure 1. A portion of the prepared nanocomposite

hydrogels with/without curcumin was lyophilized for characterization

study and loading/entrapment efficiency determination.

3.3 |Preparing niosomal emulsion

The primary emulsion is a water in oil (W/O) emulsion produced by

emulsifying the water phase containing the drug-loaded hydrogel.

Briefly, 10 ml of the prepared hydrogel was added little by little to

olive oil and span 80 as an emulsifier to prepare the first layer of

HASELI ET AL.3of18

FIGURE 1 Schematics of crosslinking between (a) chitosan and chitosan, (b) chitosan and agarose, and (c) agarose and agarose using glyoxal

as a crosslinker

4of18 HASELI ET AL.

niosomes. Then, the water phase was used to produce the second

layer and form the bi-layer vesicles, called niosomes. The hydrophobic

phase was removed from the top of the suspension by a laboratory

sampler due to the affinity of the external aqueous phase of the fabri-

cated niosomal emulsion to the hydrophilic (water) phase. The remain-

ing hydrophilic phase, which includes the fabricated niosomes, was

centrifuged at 6000 rpm for 8 min. The nanocomposite fabrication

and loading in niosomes are depicted in the graphical abstract

(Created with BioRender.com).

3.4 |Measurement of curcumin loading and

encapsulation efficiency

Curcumin loading and encapsulation (entrapment) capacity was speci-

fied to assess the effect of MMT on curcumin loading efficiency in

CS-Aga hydrogel. Lyophilized nanocarriers of CS-Aga (1 mg) and CS-

Aga-MMT loaded with curcumin (1 mg) were dispersed in 1 ml PBS,

then added 1 ml ethyl acetate. Separation of ethyl acetate was done

after stirring the mixtures. The free content of curcumin (Cur) in the

ethyl acetate phase was measured by a UV–Vis spectrophotometer at

419 nm.

The tests were performed in triplicate. Curcumin encapsu-

lation and loading efficiency were determined using Equations 1 and

52,53

respectively.

3.5 |Characterization of the prepared delivery

system

A Nanotrac wave analyzer (Microtrac, Osaka, Japan) was used to

determine nanocarriers' average size and surface charge by dynamic

light scattering (DLS) and zeta potential measurement, respectively.

The surface morphology of the nanocomposites was studied by Field

Emission Scanning Electron Microscope (FESEM) (TESCAN, MIRA III

model, Czech Republic) at an accelerated voltage of 30 kV. The exper-

iments were done at a scattering angle of 90after probe sonication

of the solution. FTIR was used to verify the presence of chitosan, aga-

rose, montmorillonite, and curcumin in the fabricated platform by

observing the change in FTIR spectra after adding each component.

The spectroscopy was done at 25C by a KBr pellet. A pellet of thick-

ness 0.1 cm was made by grinding 1 mg of each sample in a mortar

pestle with KBr. KBr pellet was used as blank. The range of scanning

was 600 to 4000 cm

1

. The spectra of CS, CS-Aga, CS-Aga-MMT,

and CS-Aga-MMT-Cur were recorded on freeze-dried samples using

a PerkinElmer Spectrum Version 10.03.06. The changes in the crys-

tallinity of samples were examined to verify the interactions

between chitosan, agarose, nanoparticles and curcumin, which lead

to covering the crystalline peaks of curcumin and forming an amor-

phous phase. The analysis was done using an X-ray diffractometer

(XRD-6100, Shimadzu, Japan) in the range of 10≤2θ≤80using a

Cu Kαsource with λ=1.54056. The step size was 0.05 degrees with

a 1-s interval between each step. XRD patterns were analyzed by

HighScore plus 3.

3.6 |Drug release analysis

Curcumin release from nanoniosomal emulsions was inspected by the

dialysis method at pH 7.4 and 5.4 to evaluate the pH-sensitive feature

of the fabricated nanocarrier and curcumin controlled-release.

One milliliter of the nanoniosomal solution was added into a dialysis

bag (Mw cutoff 12,000 g mol

1

) and immersed in 60 ml of PBS con-

taining ethanol 20% v/v.

At particular time points of 0, 12, 24, 48, 72, 96, and 120 hours,

300 μl of samples were elicited and substituted with the same volume

of fresh PBS. The released curcumin was measured by UV–Vis spec-

trophotometric analysis of the elicited buffer (Thermo Biomate

5, Thermo Scientific, US) at 419 nm. The above procedure was done

three times. Curcumin release percentage was calculated using

Equation 3:

Curcumin released %ðÞ¼Curcumin½rel

Curcumin½load 100 ð3Þ

In the above relation, [Curcumin]

load

is curcumin loaded in the

hydrogel, and [Curcumin]

rel

is curcumin released from the nanonio-

somes. The statistical analysis (ANOVA) was done based on curcumin

release percentage from CS-Aga-MMT-Cur at pH 5.4 and curcumin

release from all other groups to corroborate the pH sensitivity of the

nanocarrier.

Encapsulation Efficiency %

ðÞ

¼ðCurcumin initial amountÞðFree Curcumin in ethyl acetateÞ

ðInitial CurcuminÞð1Þ

Loading Efficiency %

ðÞ

¼ðCurcumin initial amountÞðFree Curcumin in ethyl acetateÞ

ðNanocomposite total amountÞð2Þ

HASELI ET AL.5of18

3.7 |Mathematical models of curcumin release

Curcumin release results were fitted to drug release models to deter-

mine the release mechanism in neutral and acidic conditions. The

zero-order, first-order, Higuchi, Hixson–Crowell, and Korsmeyer–

Peppas models were used for mathematic modeling. The fitting was

performed with GraphPad 7. The R squares of the above models were

calculated, and according to the best-fitting regression parameters,

the release mechanism was specified at pH 7.4 and 5.4. After fitting,

factors without statistical meaning based on a p-value less than 0.05

were expunged from models. Also, the drug release data below 60%

of the cumulative drug release were used for the Korsmeyer–Peppas

model based on the instructions for this model.

3.8 |Cell Culture

DMEM media containing 10% fetal bovine serum (FBS), penicillin

(100 U.ml

1

) and streptomycin (100 μg.ml

1

) were used to culture

cells at 37C in a humidified incubator with 5% CO

3.9 |In vitro cytotoxicity assay

The cytotoxicity of curcumin, CS-Aga-MMT nanocomposite loaded

with curcumin (CS-AGA-MMT-Cur), and nanoniosomes containing

CS-Aga-MMT without curcumin on MCF-7 cell line were studied

using MTT assay. A growth medium of 2 10

MCF-7 cells was

added to wells of a 96-well plate and incubated for one night to

achieve cell adherence. After 24 h, with 70% confluency of cells

attached to the plate, the samples at 6.4 μg.ml

1

, with regard to the

loading of curcumin (64%) in the prepared hydrogel, were incubated

with the cells to evaluate each sample's cytotoxicity. Selecting this

concentration leads to comparing equal concentrations of the samples

to study the impact of the prepared platform on cytotoxicity via

changing the release profile. The MTT assay was conducted as in the

former study.

The optical density of samples was read using an

ELISA reader at 570 nm to determine the viability of samples com-

pared with the control group's viability put on 100%. The mean and

the standard error of the mean (SEM) of cell viability for each group

were specified. GraphPad InStat 3 was used for the statistical analysis

to compare the means, standard errors, and the number of replicates

(N=3) between the groups.

3.10 |Flow cytometry

The induction of apoptosis and necrosis was analyzed after Annexin

V-FITC and Propidium Iodide (PI) double staining (BioLegend, UK). Ini-

tially, MCF-7 cells were treated with curcumin, CS-Aga-MMT-Cur

nanoniosome, and CS-Aga-MMT without curcumin for 3 days. The

concentration of all these samples was 6.4 μg.ml

1

to compare sam-

ples at an equal concentration according to the same reason

mentioned above. The fluorescence intensity of cells was determined

in FL-1 (FITC) and FL-3 (PI) channels with the same procedure con-

ducted in former studies.

29,40

Quadrant statistics were used to deter-

mine the percentage of cells in different stages of cell death in each

quadrant. The values in the upper left, upper right, lower right, and

lower left quadrants represent necrotic (Q1), late apoptotic (Q2), early

apoptotic (Q3), and viable (Q4) cells, respectively. The described assay

was conducted in triplicate to examine the enhanced apoptosis induc-

tion of nanoniosomes loaded with CS-Aga-MMT-Cur, owing to curcu-

min's sustained-release from the fabricated niosomes.

4|RESULTS AND DISCUSSION

4.1 |FTIR

The addition of all components to the fabricated nanocomposite was

ascertained via FTIR based on the observation of peaks signifying

chitosan (CS), agarose (Aga), montmorillonite (MMT), and curcumin

(Cur) interactions. In the chitosan spectrum, the 890 cm

1

peak is

attributed to the C–H bending out of the plane of the monosaccha-

ride's ring. Two bands at 1020 cm

1

and 1060 cm

1

were detected

due to C–O stretching. The band at 1149 cm

1

could be ascribed to

the asymmetric extension of the C–O–C bridge. The small peak

observed at 1272 cm

1

is due to the chitosan hydroxyl groups bend-

ing vibrations. A band at 1317 cm

1

is characteristic of the amide III

C–N stretching. The absorption band detected at 1375 cm

1

is in

agreement with former detections in the literature and is ascribed to

the CH

symmetrical deformation.

At 1413 cm

1

, the peak is

referred to as CH

bending. The band located at 1583 cm

1

corre-

sponds to C=O stretching of amide I, which confirms N-acetyl groups

presence in the acetylglucosamine unit of chitosan.

The observed

band at 2856 cm

1

could be credited to C–H stretching. O–H stretch-

ing band can be observed at 3355 cm

1

, in agreement with previous

studies.

The FTIR spectrum of CS-Aga was compared with the chit-

osan spectrum to investigate the interaction between chitosan and

agarose and confirm the addition of agarose by new observed peaks.

The FTIR spectrum of CS-Aga shows two additional peaks at 613 and

655 cm

1

that represent the C–H bending characteristic of aga-

rose.

58,59

The observed band at 890 cm

1

, which was ascribed to the

C–H bending of chitosan moved to 917 cm

1

, probably because of

alterations in molecular interactions between chitosan and agarose. A

new band at 954 cm

1

denotes the anhydrogalactose units present in

the anhydrogalactose residues of agarose and is in accordance with

the spectrum of pure chitosan reported before.

60–62

The peak

observed at 1060 cm

1

in the pure chitosan spectrum shows an

increased intensity ratio, which is ascribed to the presence of C–O

stretch in both agarose, in line with the former results in the

literature,

58,63

and in chitosan. Besides, the intensity ratio of the band

at 1150 cm

1

increased in the CS-Aga likewise. The band at

1270 cm

1

representing O–H in the chitosan spectrum was observed

again in the CS-Aga spectrum. The intensity ratio of the observed

bending peak at 1413 cm

1

and the C=O stretching peak at

6of18 HASELI ET AL.

1560 cm

1

increased in the CS-Aga as compared with the CH

because of the presence of C–C stretch in agarose with CH

bending

of chitosan at 1413 cm

1

and the O–H bending of agarose with C=O

stretching of chitosan at 1583 cm

1

. Indeed, the presence of these

peaks at the same wavenumber leads to the intensity ratio increase.

The band at 1630 cm

1

signifies the O–H bending and is in accord

with reported spectrums of agarose in the literature.

The intensity

ratio of this band also increased because of the N–H bending vibra-

tion of NH

in chitosan and O–H bending of agarose at this wave-

number.

Additional peaks like the band at 2160 cm

1

are among

the characteristic peaks of agarose, affirming agarose addition to chit-

osan.

The band at 2856 cm

1

observed in the CS spectrum was

detected in the CS-Aga. The intensity ratio of the band at 3374 cm

1

increased in comparison with CS. This increase is ascribed to the O–H

stretching vibration of agarose and O–H stretching in chitosan.

64,66

the spectrum of CS-Aga-MMT, a new band at 776 cm

1

is observed

that refers to the CH

in montmorillonite.

The band denoting C–H

at 890 cm

1

detected in the FTIR of pure chitosan was detected again

in CS-Aga-MMT. Also, the band attributed to anhydrogalactose resi-

dues of agarose in CS-Aga was observed in the CS-Aga-MMT spec-

trum at 930 cm

1

With regard to the CS-Aga spectrum, the intensity ratio of the

band at 1150 cm

1

increased since montmorillonite has Si–O stretch-

ing and agarose has C–O stretch at the same wavenumber.

The

peaks at 1413 cm

1

and 1583 cm

1

were observed again in the CS-

Aga-MMT spectrum. Due to the addition of MMT nanoparticles con-

taining H–O–H bending to CS-Aga hydrogel, the band at 1629 cm

1

shows an increased intensity ratio.

The band at 2856 cm

1

was

detected again in the CS-Aga-MMT spectrum.

In the spectrum of CS-Aga-MMT-Cur, the band at 930 cm

1

shifted to 925 cm

1

with a reduction in intensity ratio that indicates

probable electrostatic interactions between curcumin and hydrogel

polymers. These interactions lead to the enhanced complexation of

components in the nanocomposite. The band at 1150 cm

1

decreased

in the intensity ratio due to C–O–C interaction in curcumin with other

components.

The bands at 1413 cm

1

and 1560 cm

1

were

detected again with a reduced intensity ratio compared with former

spectrums, mainly due to the complexation with curcumin. The bands

at 2918 cm

1

and 3371 cm

1

ascribed to C–H stretch and O–H

stretch, respectively, are characteristic peaks of curcumin detected

with reduced intensity ratio due to the complexation with other com-

ponents and are in accord with the spectrum of curcumin.

70,71

The

existence of the characteristic peaks verifies the addition of chitosan,

agarose, and montmorillonite to the fabricated nanocomposite.

Figure 2represents the FTIR spectra of samples.

4.2 |XRD analysis

XRD patterns of samples were used to determine crystalline structure

alterations by the addition of components to the nanocomposite.

XRD spectra for CS, CS-Aga, CS-Aga-MMT, and CS-Aga-MMT-Cur

were taken, as displayed in Figure 3. The diffractogram of CS shows

two characteristic peaks at 2θequal to 14.5 and 20.78 θ, which are

representative of crystalline chitosan, in agreement with the litera-

ture's results.

The XRD pattern of the CS-Aga sample indicates that

the crystalline peak in the spectrum of chitosan (2θ=20.78θ) has

become broader, with a reduced intensity ratio, and shifted to the

right, at 22.57θ. This reduction can be ascribed to the addition of

agarose to chitosan. Agarose has a broad peak around 18–29 θ.

The addition of agarose to chitosan results in a semi-crystalline struc-

ture, which is displayed in the spectrum with a reduction in intensity

ratio and the formation of a broad hump (2θ=22.57θ) compared

with pure chitosan. The reduction in the intensity ratio of crystalline

chitosan was also observed after crosslinking in a study by Julkapli

et al

In the XRD spectrum of CS-Aga-MMT, broad peaks were

observed at 12.01θand 20.78θ. The peak observed at 12.1 θis a

characteristic of montmorillonite (JCPDS card no. 13-0135), showing

FIGURE 2 FTIR spectra of

the samples

HASELI ET AL.7of18

its crystalline structure, and complies with the reported results by

Samudrala et al

The reduction in the intensity ratio of this peak and

its broadening demonstrates the successful complexation of crystal-

line montmorillonite with other components and the formation of an

amorphous phase. Moreover, the peak at 28.51θin CS-Aga-MMT is

another characteristic peak of montmorillonite, which verifies the

incorporation of MMT in CS-Aga hydrogel.

74,75

In the CS-Aga-MMT-

Cur pattern, the intensity ratio of the peak at 20.78θincreased in

comparison with CS-Aga-MMT. This increase is due to the crystalline

peaks of curcumin between 5and 30and confirms the addition of

curcumin with regard to characteristic peaks of curcumin.

The suc-

cessful incorporation of curcumin in the nanocomposite with an amor-

phous structure was verified as the XRD pattern of CS-Aga-MMT-Cur

displays no crystalline peak attributed to curcumin between 20and

30.

Besides, the FTIR results confirmed the loading of Cur in the

nanocomposite according to the interactions observed in FTIR after

the addition of curcumin.

4.3 |Morphology observation

Lyophilized nanocomposite hydrogels that incorporated curcumin

were observed by FESEM (Figure 4). According to the images, the

spherical shape of nanocomposites is evident. The spherical shape is

an apt shape of nanocarriers for drug delivery applications.

From

the homogeneous surface on the nanocomposites, good compatibility

between its components was patent. According to measurements

conducted by ImageJ software, the average size of the nanocompo-

sites was 61.5 nm.

4.4 |DLS and Zeta potential

Polydispersity and nanoniosomes size were measured by DLS. The

niosome size distribution was around 400 nm with a PDI of 0.3

(Figure 5), which was sonicated to reduce their size to 241 nm. This

size distribution is satisfactory for applying niosomes in drug delivery

and is in the range of niosomal platforms fabricated before.

78–80

Addi-

tionally, zeta potential can verify nanocarriers' stability. The zeta

potential was +47 mV for the nanocarriers demonstrating good stabil-

ity (Figure 6).

Also, the positive charge of niosomes improves their

interaction with cell membranes.

4.5 |Effect of montmorillonite nanoparticles on

loading and encapsulation efficiency

Poor solubility of anti-cancer agents is a prevalent challenge.

29,30,40,83

The bioavailability of curcumin has been widely reported to be very

FIGURE 3 XRD patterns of

the samples

FIGURE 4 FESEM of lyophilized nanocomposite hydrogels of CS-

Aga-MMT loaded with curcumin

8of18 HASELI ET AL.

poor as a consequence of its low solubility (1 μg.ml

1

16,17

In this

regard, improving curcumin's loading efficiency is a challenge. The

encapsulation and loading efficiencies of curcumin in CS-Aga-Cur and

CS-Aga-MMT-Cur were determined by Equations 1and 2, respec-

tively, to study MMT nanoparticles' effect on loading and entrapment

efficiency. The loading efficiency for CS-Aga-Cur was 63%, which

increased to 76% in CS-Aga-MMT-Cur. This enhanced loading is

ascribed to the role of MMT nanoparticles. In fact, the siloxane group

in montmorillonite interacts with chitosan amine groups and hydroxyl

groups in agarose and curcumin to form hydrogen bonds and create a

high interpenetrating network. The interlocked structure of the nano-

composite leads to the model drug entrapment within the structure.

The FTIR spectrum indicated these interactions between the curcumin

and MMT nanoparticles when the drug was loaded into the hydrogel

containing MMT nanoparticles. Besides, the introduction of MMT into

CS-Aga hydrogel leads to the provision of a higher surface for

interactions between hydrogel polymers (chitosan and agarose),

montmorillonite, and the model drug (curcumin). The loading of cur-

cuminachievedinthisstudywashigherthanthepH-responsive

platforms developed before. Ahmadi Nasab et al. developed pH-

responsive chitosan mesoporous silica nanoparticles to treat glio-

blastoma cancer. The loading of curcumin in this drug delivery sys-

tem was 8.81%.

In another study, solid lipid nanoparticles (SLN)

were synthesized to deliver curcumin. The loading efficiency of cur-

cumin was 71%.

The loading efficiency of saponin-coated curcu-

min nanoparticles was 15%.

The average curcumin loading

efficiency in diblock copolymer micelles developed for the treatment

of breast cancer was 5.97%.

Also, Li et al. fabricated thiolated

chitosan-coated liposomal hydrogels as curcumin carriers to prevent

breast cancer recurrence. The loading efficiency of curcumin was

reported 3.96%.

Also, the entrapment efficiency of curcumin in the chitosan-

agarose hydrogel without/ with MMT was 77% and 92%, respectively.

Similarly, the entrapment percentage in this study was higher than

curcumin encapsulation efficiency in saponin-coated curcumin

nanoparticles,

thiolated chitosan-coated liposomal hydrogels,

and

poly (lactic-co-glycolic) acid nanoparticles (PLGA NPs) loaded with

curcumin.

Table. 1recapitulates the efficiencies before and after the

addition of montmorillonite. Also, Table 2summarizes the loading or

encapsulation efficiencies of other curcumin delivery platforms.

100

0 100 200 300 400 500 600 700 800 900 1000

Frequency (%)

Diameter (nm)

FIGURE 5 Size distribution of

CS-Aga-MMT-Cur nanocarrier

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 102030405060

Intensity (a.u)

Zeta Potential (mV)

FIGURE 6 Zeta potential for

the fabricated CS-Aga-MMT-Cur

nanocarrier

TABLE 1 Alteration of loading and entrapment after the addition

of montmorillonite (MMT)

CS-Aga CS-Aga-MMT Effect of MMT (%)

Loading (%) 63 76 +13

Entrapment (%) 77 92 +15

HASELI ET AL.9of18

4.6 |Curcumin release from the nanocarriers

The release profile was studied to confirm the sustained and pH-

sensitive release of curcumin from the prepared platform. As men-

tioned above, the release of curcumin was assessed by dialysis

method at pH 7.4 and 5.4 at 37C for a period of 120 h (Figure 7). As

illustrated in Figure 7, the cumulative release of curcumin from CS-

Aga-MMT-Cur and CS-Aga-Cur at pH 7.4 during 24 h was 24% and

30%, respectively. In contrast, the cumulative release during 24 h at

pH 5.4 (acidic conditions) was 67% for CS-Aga-MMT-Cur and 48%

for CS-Aga-Cur, respectively. At pH 7.4, the cumulative release from

CS-Aga-MMT-Cur is less than CS-Aga-Cur because of the impact of

MMT nanoparticles on the release behavior. The interactions of func-

tional groups in polymers like amine groups in chitosan and hydroxyl

groups in agarose and curcumin with the oxygen of siloxane group in

montmorillonite form hydrogen bonds between components and keep

the structure of the nanocomposite intact, which results in reduced

cumulative release at pH 7.4 in comparison with CS-Aga-Cur. At

pH 5.4, 98% of curcumin was released after 120 h, while at pH 7.4,

only 73% was released after 120 h. Moreover, a gradual release of

curcumin was observed prior to the sustained-release at pH 7.4,

which can be observed in Figure 7.

The observed protracted-release under neutral conditions

enables the nanocarrier to retain the drug and release more drug at

the tumor site over a long period. The release behavior of the groups

containing montmorillonite at pH 5.4 and 7.4 express the pH-

sensitive release from the prepared nanocarriers. This feature is

attributed to the dissolution of MMT nanoparticles and the release

of Al and Si ions, along with the pH-responsive property of chito-

san.

Indeed, the protonation of the chitosan amine group and

hydroxyl groups in agarose and curcumin, along with the release of

ions, provides repulsive forces between nanocomposite components,

which lead to the increased release under acidic conditions.

There-

fore, the increase in the drug release rate for CS-Aga-MMT-Cur at

pH 5.4 than at pH 7.4 can be attributed to the MMT nanoparticles'

pH-responsiveness and protonation of polymers in low pH.

46,92

There was a significant difference between pH 5.4 with montmoril-

lonite (MMT) as the control and all other groups with p< 0.001, as

presented in Figure 8.

Besides, the enhanced sustained release by niosomes can be

attributed to the oil phase in the niosomes, which functions as a

membrane to protract the release period after the nanocomposite

dissolution. A similar outcome was achieved in the fabrication of a

Chitosan-based platform.

Hence, the niosomal emulsion stabilizes

the nanocarrier at pH 7.4 and protracts the release from the prepared

pH-responsive hydrogel at pH 5.4.

The impact of niosomal emul-

sions on protracting the release period is manifest compared with the

release behavior of curcumin-loaded CS-Aga-MMT nanocomposite

fabricated in our former study and liposomal hydrogels fabricated by

Li et al

50,89

At pH 7.4, 31.57% of curcumin was released from liposo-

mal hydrogels after 12 h, while in the current study, only 15% of

curcumin was released at pH 7.4 after 12. Ju et al. prepared pH-

responsive liposomes for curcumin delivery, which released 35.25% of

curcumin at pH 7.4. after 24 h. Curcumin release from CS-Aga-MMT

nanocarrier is 24% after 24 h at pH 7.4. Also, the cumulative release

from the fabricated nanocarriers after 24 h at pH 5.4 is less than

reported by Ju et al

Also, in the current study, 67% of curcumin was

released at pH 5.4 after 24 h, whereas 60% of curcumin was released

from iron oxide nanoparticles loaded with curcumin after 8 h at

pH 5.

The simultaneous improvement of the loading and entrapment

efficiency of curcumin in niosomes and attaining sustained drug

TABLE 2 Encapsulation and loading efficiency of curcumin in other platforms

Curcumin delivery platforms Loading % Encapsulation % Reference

pH-responsive chitosan mesoporous silica

nanoparticles

8.81 —85

Solid lipid nanoparticles (SLN) 71 —86

Saponin-coated curcumin nanoparticles 15 91 87

Diblock copolymer micelles 5.97 64 88

Thiolated chitosan-coated liposomal hydrogels 3.96 88.75 89

Curcumin incorporated in montmorillonite (MMT) —67 91

Curcumin delivery by PLGA nanoparticles 75 —90

024487296120

100

time(h)

Cumulative Release % of Curcumin

pH = 5.4 without MMT

pH = 5.4 with MMT

pH = 7.4 without MMT

pH = 7.4 with MMT

FIGURE 7 Cumulative release of curcumin from nanocarriers

encapsulating chitosan-agarose hydrogels with/without

montmorillonite (MMT) at acidic and neutral conditions

10 of 18 HASELI ET AL.

release is evident when the release profile of the current platform is

compared with former studies. In a study conducted by Xu et al., the

entrapment efficiency of curcumin was 92%, whereas 80% of curcu-

min was released after 24 h.

In contrast, this study improved the

entrapment efficiency and prolonged the release period. In another

study, optimizing the formulation of niosomes for curcumin delivery

resulted in the entrapment efficiency similar to this study but compar-

ing the release of curcumin shows the current platform has better sus-

tained release.

4.7 |Mathematical models of drug release

By fitting the drug release data to mathematical models of drug

release, the mechanism of drug release was determined.

In neutral

and acidic pH, the drug release data were fitted to zero-order, first-

order, Higuchi, Hixson-Crowell, and the Korsmeyer-Peppas models to

determine the probable alterations in the release mechanism by pH

change. Analyzing the above models exhibited the Korsmeyer–Peppas

model best fits the release data at pH 7.4.

The Korsmeyer–Peppas model is used to examine drug release

when the release mechanisms are complicated and involve more than

one mechanism. In the Korsmeyer–Peppas model, n is the diffusional

exponent demonstrating the release mechanism. The value of

0.43 < n< 0.85 in this model designates non-fickian (or anomalous

transport) release at pH 7.4. In this case, the release is controlled by

diffusion and erosion of the polymeric matrix according to the model

and previous studies.

97,98

The same kinetics was observed in the

release of curcumin from poly (lactic-co-glycolic) acid nanofibers fabri-

cated by Sampath et al

The time-dependent anomalous transport

occurs from the rearrangement of polymeric chains (dissolution) and

diffusion simultaneously.

100

Also, the Higuchi model describes

the release mechanism, including both diffusion and dissolution.

101

The better fitting of the release data to the Higuchi model after the

Korsmeyer–Peppas at pH 7.4 is because the Higuchi model represents

simultaneous dissolution and diffusion, which is the same as the

mechanism represented by the Korsmeyer–Peppas model based on

the n value in this study.

Additionally, the same analysis was performed for pH 5.4. The

values showed that the Korsmeyer–Peppas model best fits

the release data (Figure 10) at pH 5.4. According to this model, the

release mechanism is Super Case II for n> 0.85. In this mechanism,

the dissolution of the polymeric matrix occurs much faster than dif-

fusion. The reason for this faster dissolution is the protonation of

components under acidic conditions, which produces repulsive

forces between components. Also, the alteration of the mechanism

governing the model drug's release represents the prepared nanocar-

rier's pH-responsive feature. The mathematical analysis at pH 7.4

andpH5.4isillustratedinFigure9. The modeling results are recapit-

ulated in Table. 3.

4.8 |In vitro cytotoxicity study

MTT assay was used to determine the fabricated curcumin-loaded

nanocarriers' cytotoxicity and to find out if the fabricated platform for

curcumin delivery can be applied with substantial cytotoxicity in com-

parison with free curcumin. The MTT assay measured the cytotoxic

effects of CS-Aga-MMT-Cur nanocarriers and free curcumin on

MCF-7 cells after 72 hours of incubation (Figure 10). Cytotoxicity of

cells treated with CS-Aga-MMT not loaded with curcumin was also

assessed to evaluate the unloaded nanocarriers' effect on the viability

of cells. According to results represented in Figure 10, cell viability in

the CS-Aga-MMT group that was not loaded with curcumin was not

significantly reduced compared with the control group. This result

demonstrates that the fabricated nanocarrier does not have cytotoxic

effects on cancer cells by itself and only improves the release behav-

ior, as discussed before. A significant decrease in viability was

120

100

time(h)

CurcuminC

umulativeRelease%

pH=5.4 without MMT

pH=5.4 with MMT

pH=7.4 without MMT

pH=7.4 with MMT

***

*** *** ***

*p<0.05

** p<0.01

*** p<0.001

Com

arison with

H=5.4 with MMT

***

FIGURE 8 Cumulative release

percent of curcumin at each time point for

each group. Difference between pH 5.4

with MMT and all other groups is

significant at p< 0.001(***)

HASELI ET AL.11 of 18

observed in the group treated with free curcumin compared with the

control group (p< 0.001). The same result was observed for the CS-

Aga-MMT-Cur compared with the control group. In addition, the via-

bility of the group treated with CS-Aga-MMT-Cur decreased with

p< 0.001 compared with free curcumin. As mentioned before, curcu-

min is a highly hydrophobic drug with poor solubility.

102

Thus, the

effective delivery of free curcumin is hindered due to its poor solubil-

ity and instability in the cell medium. By incorporating curcumin in the

CS-Aga-MMT nanocarrier, the prolonged-release behavior leads to

the longer presence of soluble curcumin in the tumor cells' environ-

ment, causing higher cytotoxicity.

4.9 |Flow cytometry study

Apoptosis induction in breast cancer cell lines, including MCF-7, is

based on triggering a set of reactions that lead to cell membrane per-

meability, followed by swelling and potential loss in the membrane.

103

Several pathways have been identified for promoting apoptosis in can-

cer cells with curcumin. Regulation of the NF-κB signaling pathway,

104

targeting enzymes involved in the production of reactive oxygen spe-

cies (ROS),

8,12

down-regulation of bcl2 as an antiapoptotic gene,

and

up-regulation of bax and p53 genes are among the pathways proposed

for apoptosis induction on MCF-7 cells by curcumin.

105

pH 5.4

pH 7.4

Zero-Order

First-Order

Higuchi

Hixon-Crowell

sro

K -Peppas

y = 0.6535x

R = 0.9826

100

0 20 40 60 80 100 120

Cumulative Drug

Release %

Time (h)

y = 1.0724x

R = 0.8813

100

0 20 40 60 80 100 120

Cumulative Drug Release

Time (h)

y = -0.0333x + 4.3861

R = 0.9777

0 20 40 60 80 100 120

Ln ( Non-released

percentage of drug)

Time (h)

y = -0.0128x + 4.6514

R = 0.9727

0 20 40 60 80 100 120

Ln ( Non-released

percentage of drug)

Time (h)

y = 10.46x

R = 0.9801

100

0 5 10 15

cumulative release

percentage

sqrt Time (h)

y = 6.0712x

R = 0.9914

100

0 5 10 15

Cumulative release

percentage

sqrt Time (h)

y = -0.0272x + 4.1186

R = 0.8974

0 20 40 60 80 100 120

Cubic root of non-

released percentage

Time (h)

y = -0.016x + 4.577

R = 0.9088

0 20 40 60 80 100 120

Cubic root of non-

released percentage

Time (h)

y = 1.0087x

R = 0.9988

0246

Log (Cumulative

release percentage)

Log (Time(h))

y = 0.7778x

R = 0.9998

0246

Log( Cumulative

release percentage)

Log (Time(h))

FIGURE 9 Drug release

modeling

12 of 18 HASELI ET AL.

The mitochondrial-dependent pathway is another pathway

for apoptosis induction in cancer cells by curcumin. In this path-

way, cytochrome C is released from cells, which leads to a

caspase-9-caspase-3 cascade. Subsequently, poly (ADP-ribose)

polymerase (PARP) by the ‘effector’caspases-3 acts as a cleav-

ing agent to fragment DNA leading to cell disintegration and

apoptosis.

106,107

Externalization of phosphatidylserine (PS) from the cell's inner

membrane to its outer layer is a prominent feature of apoptosis induc-

tion. The annexin-V protein binds to PS specifically, and fluorescent

labeling of annexin-V allows flow cytometric detection of externalized

PS and, as a result, apoptotic cells. Since the loss of membrane perme-

ability in which PS externalization occurs is regarded as the onset of

apoptosis, early apoptotic cells are known as Annexin V-positive.

108

the early apoptosis stage, cell membrane integrity is not totally com-

promised; thus, PI cannot enter the cells making early apoptotic

cells PI-negative (Annexin V-FITC

/PI



). However, in the late apo-

ptosis stage, cell membrane degradation allows PI to enter the cells

making late apoptotic cells Annexin V-positive/ PI-double-positive

(Annexin V-FITC

/PI

109

Dual staining with Annexin V and PI

enables discriminating apoptotic and necrotic cell death since

necrotic cells are Annexin V-negative/PI-positive (Annexin V-

FITC



/PI

110

The apoptosis assay was conducted to discover the effect of the

sustained-release of curcumin achieved by the application of MMT

nanoparticles in hydrogel and niosomal emulsions on curcumin's anti-

cancer activity. As can be seen in Figure 11, the apoptotic and

necrotic induction impact of CS-Aga-MMT-Cur, free curcumin, and

blank CS-Aga-MMT (without drug) was studied by using Annexin

V-FITC and PI double staining.

The percentage of viable cells (Q4) in blank CS-Aga-MMT did not

decrease significantly compared with the viable cells in control, com-

plying with the cytotoxicity analysis mentioned earlier. Also, viable

cells treated with free curcumin (26.9%) were more than viable cells

observed in CS-Aga-MMT-Cur (9.36%), which agrees with the cyto-

toxicity results. The percentage of necrotic cells (Q1) for free curcu-

min was 62.1%, while necrotic cells in CS-Aga-MMT-Cur was 20.7%.

This difference is due to the direct exposure of cells to free curcumin.

Besides, the apoptotic cells (early and late) increased in CS-Aga-

MMT-Cur compared with free curcumin. This outcome reflects the

enhanced cytotoxicity of curcumin via increased apoptosis achieved

by curcumin sustained-release. Therefore, the prolonged-release in

the tumor microenvironment (pH 5.4), as illustrated before, is due to

the copolymer and crosslinked structure of the CS-Aga matrix and the

interaction between drug and MMT nanoparticles and niosomal emul-

sion, which lead to the protracted release of the drug and increased

apoptosis.

***$$$

***

100

Control CS-Aga-MMT CS-Aga-MMT-Cur Cur

Viability %

FIGURE 10 Cytotoxicity of curcumin, CS-Aga-MMT nanocarrier

loaded with curcumin, and CS-Aga-MMT on MCF-7 cells after 72 h

incubation. Each experiment was conducted three times in duplicate

to obtain mean ± SEM. The viability of free curcumin and CS-Aga-

MMT-Cur is significantly different from the control group (p< 0.001

(***)). Also, the viability of CS-Aga-MMT-Cur is considerably less than

free curcumin with p< 0.001 ($$$)

TABLE 3 Equations used to fit models to release data

Model Equation R

pH 7.4 Zero-order C

=1.0724 t 0.8813

First-order ln 1Mt

M∞



¼4:6514 0:0128t0.9727

Higuchi Q¼6:0712t1

20.9914

Hixon-Crowell 1Mt

M∞



3¼4:577 0:016t0.9088

Korsmeyer–Peppas ln Mt

M∞



¼0:7778ln t 0.9998

pH 5.4 Zero-order C

=0.6535 t 0.9828

First-order ln 1Mt

M∞



¼4:3861 0:0333t0.9777

Higuchi Q¼10:46t1

20.9801

Hixon-Crowell 1Mt

M∞



3¼4:1186 0:0272t0.8962

Korsmeyer–Peppas ln Mt

M∞



¼1:0087ln t 0.9988

HASELI ET AL.13 of 18

5|CONCLUSION

Although curcumin shows promising anti-cancer properties, its

insoluble and unstable nature is a challenge for using it as a free

drug for cancer therapy during a long period of time. To this end,

fabricating a delivery system with stimuli-sensitive properties for

targeted delivery, improved loading capacity, and prolonging its

release period is indispensable. In this study, pH-responsive nano-

compositesweredevelopedtoattainbothenhancedloadingand

sustained-release of curcumin. Incorporation of MMT nanoparticles

into nanocomposites improves loading efficiency up to 76% and

provides pH-responsive release due to the interaction with hydro-

gel polymers. The addition of nanoparticles to the hydrogel matrix

provides interactions between curcumin and the polymers to pre-

servecurcuminatpH7.4andreleaseitintheacidicconditionof

cancer cells. Besides, the encapsulation of nanocomposites into

niosomes extended the release interval by the oil phase of the nio-

somal emulsion to further prolong the release period. In addition,

nanocarriers prepared by this method improved the apoptosis

activity of curcumin and reduced cancer cells viability in compari-

son with curcumin-treated cells. Accordingly, the current fabricated

platformcanbeemployedasaprospectivepH-responsive

nanocarrier which can be employed for curcumin delivery and ame-

liorate the mentioned shortcomings assigned to curcumin for can-

cer therapy.

AUTHOR CONTRIBUTIONS

Shabnam Haseli: Formal analysis (equal); investigation (equal);

methodology (equal). mehrab pourmadadi: Formal analysis (equal);

funding acquisition (equal); methodology (equal). Amirmasoud

Samadi: Conceptualization (equal); methodology (equal); writing –

original draft (equal). Fatemeh Yazdian: Project administration

(equal); supervision (equal); validation (equal). Majid Abdouss: For-

mal analysis (equal); investigation (equal). Hamid Rashedi: Concep-

tualization (equal); data curation (equal); supervision (equal). Mona

Navaei-Nigjeh: Formal analysis (equal); investigation (equal); super-

vision (equal).

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

PEER REVIEW

The peer review history for this article is available at https://publons.

com/publon/10.1002/btpr.3280.

FIGURE 11 Apoptosis and

necrosis induction in MCF-7 cells

by free curcumin, CS-Aga-MMT-

Cur, and blank CS-Aga-MMT. The

values in the upper left, upper

right, lower right, and lower left

quadrants represent necrotic

(Q1), late apoptotic (Q2), early

apoptotic (Q3), and viable

(Q4) cells, respectively

14 of 18 HASELI ET AL.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the

corresponding author upon reasonable request.

ORCID

Fatemeh Yazdian https://orcid.org/0000-0002-0550-9821

Hamid Rashedi https://orcid.org/0000-0002-8460-0841

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How to cite this article: Haseli S, Pourmadadi M, Samadi A,

et al. A novel pH-responsive nanoniosomal emulsion for

sustained release of curcumin from a chitosan-based

nanocarrier: Emphasis on the concurrent improvement of

loading, sustained release, and apoptosis induction. Biotechnol.

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18 of 18 HASELI ET AL.

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In the present study, we present a pyranopyrazole-TiO2 which is encapsulated with a niosome as nanocarrier for delivery of curcumin into breast cancer cells. Nanocarrier porous TiO2 is biocompatible and with a high specific surface area and a large pore volume and was used to carry pyranopyrazole, which has been reported as an anti-cancer. Niosome in the outer layer, helpful for loading curcumin into the niosomal layer, demonstrates a pH-dependent release and can be effective for cancer treatment. Entrapment efficiency of curcumin was found at 81.02% in carriers. The results of MTT and flow cytometry revealed that apoptosis is notably enhanced by loading curcumin on pyranopyrazole-TiO2@niosome. Also, there was high biocompatibility with MCF-10A, while exhibiting significant anti-cancer and anti-metastatic effects on MCF-7, whose cell viability was 38.79% in the loaded curcumin on carrier and was more than other samples even, than free curcumin (42.82%). Furthermore, the regulation of gene expression in cancer cells decreased the regulation of MMP-2 and MMP-9 genes and increased the expression of caspase-3 and caspase-9 genes. Finally, fluorescence activity in MCF-7 significantly increased after treatment with samples. Graphical Abstract

Improving Curcumin Constraints with pH-Responsive Chitosan Based Graphene Oxide/Montmorillonite Nanohybrid Modified Agarose in Breast Cancer Therapy

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Nanoparticles have promisingly served as therapeutic agents in drug delivery systems to supress side effects while improving the effectiveness of treatment to deadly cancers. The present study aims to fabricate chitosan/agarose/graphene oxide/montmorillonite nanocomposites to serve as drug carriers for the prolonged release of curcumin to suppress MCF7 breast cancer cells. Offering a range of advantages, including biocompatibility and pH-sensitivity, the nanocomposites were synthesized via a water-in-oil-in-water (W/O/W) emulsification technique. Characterization studies were conducted with FTIR and XRD analyses. Dynamic light scattering (DLS) analysis was conducted to measure the mean size of the produced nanoscale emulsions, with zeta potential assessments further performed to characterize the nanocomposites in terms of surface charge. FE-SEM imagery indicated the uniform and flat surface of the synthesized nanocomposites. MTT assay results showed that the nanocomposites were most toxic to MCF7 breast cancer cells. The occurrence of apoptosis to MCF7 breast cancer cells was evident from the flow cytometry findings. Implementing a dialysis procedure, the curcumin was found to be released at a higher rate in acidic media. Altogether, our findings proved that the designed CS/AG/GO/MMT@CUR nanocomposites could be a drug carrier for treating breast cancer cells. Graphical Abstract

A Review of Novel Nanosystems in Enhancing Cell Death in Breast Cancer: Important Conclusions From Recent Advances

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Nanotechnology-based drug delivery systems can reduce side effects, increase drug release time, and control drug release. Polymeric nanoparticles used as drug nanocarriers increase drug solubility and stability. Growing concerns about the safety of chemicals and additives have necessitated the use of bioactive substances. Drug concentration is an important factor in cytotoxicity studies as it can play a role in cell death and growth inhibition. Thus, beneficial substances released by nanocarriers remove negative and unstable substances from cells. Nanocarriers are designed for prolonged release of drugs at the cancer site, resulting in potent cytotoxicity. In the most recent study, nanocarriers are prepared by double emulsion methods that stabilize and fix the shape of the nanoparticles and also prevent the nanoparticles from accelerating during drug release in vivo. In this study, Cytotoxicity investigated the effect of a newly developed nanocarrier on anti-inflammatory drug delivery to breast cancer cells(MCF-7).

Antioxidant curcumin induces oxidative stress to kill tumor cells (Review)

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Curcumin is a plant polyphenol in turmeric root and a potent antioxidant. It binds to antioxidant response elements for gene regulation by nuclear factor erythroid 2-related factor 2, thereby suppressing reactive oxygen species (ROS) and exerting anti-inflammatory, anti-infective and other pharmacological effects. Of note, curcumin induces oxidative stress in tumors. It binds to several enzymes in tumors, such as carbonyl reductases, glutathione S-transferase P1 and nicotinamide adenine dinucleotide phosphate to induce mitochondrial damage, increase ROS production and ultimately induce tumor cell death. However, the instability and poor pharmacokinetic profile of curcumin in vivo limit its clinical application. Therefore, the effects of curcumin in vivo may be enhanced through its combination with drugs, derivative development and nanocarriers. In the present review, the mechanisms of curcumin that induce tumor cell death through oxidative stress are discussed. In addition, the methods used to enhance the antitumor activity of curcumin are described. Finally, the existing knowledge on the functions of curcumin in tumors, particularly in terms of oxidative stress, are summarized to facilitate future curcumin research.

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Cancer disease is one of the most frequent life-threatening, with a high fatality rate worldwide. However, recent immunotherapy studies in various tumours have yielded unsatisfactory outcomes, with just a few individuals experiencing long-term responses. To overcome these issues, nowadays internal stimuli-responsive nanocarriers have been widely exploited to transport a wide range of active substances, including peptides, genes and medicines. These nanosystems could be chemically adjusted to produce target-based drug release at the target location, minimizing pathological and physiological difficulties while increasing therapeutic efficiency. This review highlights the various types of internal stimuli-responsive nanocarriers and applications in cancer diagnosis. This study can provide inspiration and impetus for exploiting more promising internal stimuli-responsive nanosystems for drug delivery.

Current advances in niosomes applications for drug delivery and cancer treatment

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The advent of nanotechnology has led to an increased interest in nanocarriers as a drug delivery system that is efficient and safe. There have been many studies addressing nano-scale vesicular systems such as liposomes and niosome is a newer generation of vesicular nanocarriers. The niosomes provide a multilamellar carrier for lipophilic and hydrophilic bioactive substances in the self-assembled vesicle, which are composed of non-ionic surfactants in conjunction with cholesterol or other amphiphilic molecules. These non-ionic surfactant vesicles, simply known as niosomes, can be utilized in a wide variety of technological applications. As an alternative to liposomes, niosomes are considered more chemically and physically stable. The methods for preparing niosomes are more economic. Many reports have discussed niosomes in terms of their physicochemical properties and applications as drug delivery systems. As drug carriers, nano-sized niosomes expand the horizons of pharmacokinetics, decreasing toxicity, enhancing drug solvability and bioavailability. In this review, we review the components and fabrication methods of niosomes, as well as their functionalization, characterization, administration routes, and applications in cancer gene delivery, and natural product delivery. We also discuss the limitations and challenges in the development of niosomes, and provide the future perspective of niosomes.

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Curcumin, a well-known dietary pigment derived from Curcuma longa, inhibited growth of several types of malignant cells both in vivo and in vitro. However, its mechanism of action still remains unclear. In this study, we have focused primarily on the cytotoxic effects of curcumin on three human tumor cell lines and rat primary hepatocytes. Curcumin induced apoptosis in MCF-7, MDAMB, and HepG2 cells in a dose-dependent and time-dependent manner. Apoptosis was mediated through the generation of reactive oxygen species. Attempts were made to establish the role played by endogenous glutathione on the apoptotic activity of curcumin. Depletion of glutathione by buthionine sulfoximine resulted in the increased generation of reactive oxygen species, thereby further sensitizing the cells to curcumin. Interestingly, curcumin had no effect on normal rat hepatocytes, which showed no superoxide generation and therefore no cell death. These observations suggest that curcumin, a molecule with varied actions, could be developed into an effective chemopreventive and chemotherapeutic agent.

Apoptosis Induction, Cell Cycle Arrest and Anti-Cancer Potential of Tamoxifen-Curcumin Loaded Niosomes Against MCF-7 Cancer Cells

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Background & objective: Breast cancer is the most common cancer among women. One of the most effective treatments for breast cancer is chemotherapy, in which specific drugs destroy the mass and its proliferation is inhibited. Chemotherapy is the most effective adjunctive therapy when multiple medications are used concurrently. Also, combining the drugs with nanocarrier has become an important strategy in targeted therapy. This study is designed to assess the apoptosis induction, cell cycle arrest, and anti-cancer potential of Tamoxifen-Curcumin-loaded niosomes against MCF-7 Cancer Cells. Methods: A novel niosomal formulation of tamoxifen-curcumin with Span 80 and lipid to drug ratio of 20 was employed. The MCF-7 cells were cultured and then treated with IC50 value of tamoxifen-curcumin-loaded niosomes, the combination of tamoxifen and curcumin, tamoxifen, and curcumin alone. Flow cytometry, Real-Time PCR, and cell cycle analysis tests were conducted to evaluate the induction of apoptosis. Results: Drug-loaded niosomes caused up-regulation of bax and p53 genes and down-regulation of bcl2 gene. Flow cytometry studies showed that niosomes containing tamoxifen-curcumin increased apoptosis rate in MCF-7 cells compared to the combination of tamoxifen and curcumin owing to the synergistic effect between the two drugs along with higher cell uptake by formulation niosomal. These results were also confirmed by cell cycle analysis. Conclusion: Co-delivery of curcumin and tamoxifen using optimized niosomal formulation revealed that at acidic pH of MCF-7 cancer cells, released drugs from niosomal carriers would be more effective than physiological pH. This feature of niosomal nanoparticles can reduce the side effects of drugs in normal cells. Niosomal nanoparticles might be used as a biological anti-cancer factor in treatment of breast cancer.

An electrochemical aptasensor for detection of Prostate specific antigen based on carbon quantum dots‐gold nanoparticles

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In this work, an electrochemical aptasensor was described for determination of Prostate specific antigen (PSA). Aptamer chains were decorated on the surface of a glassy carbon electrode (GCE) via carbon quantum dots/Au nanoparticles (Au/CQD). Structural analysis that used to characterize the prepared materials shows that Au/CQD nanoparticles synthesized spherically with an average size of 70 nm. Furthermore, the combination of Au nanoparticles with CQD caused the formation of crystallinity in the structure of the Au/CQD composite. For study the electrochemical performance of prepared aptasensor, Cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) were used. The results show that the aptasensor has a good selectivity to PSA over other biomaterials with the time optimized about 30 min. K4 [Fe (CN)6] was used as an electrochemical probe with the limit of detection about 2 fg.ml–1. To avoid the hazardous nature of K4 [Fe (CN)6], label based aptasensor prepared using methylene blue as an electrochemical signal producer. They provide the capability of electrochemical detection in buffer phosphate solution with high sensitivity. This article is protected by copyright. All rights reserved

An electrochemical aptasensor for detection of prostate‐specific antigen using reduced graphene gold nano composite and Cu/ carbon quantum dots

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We report a label‐free electrochemical aptamer‐based biosensor for the detection of human prostate‐specific antigen (PSA). The thiolate DNA aptamer against PSA was conjugated to the reduced graphene oxide/Au (RGO‐Au) nanocomposite through the self‐assembly of Au‐S groups. Owing to the large volume to surface ratio, the RGO‐Au nanocomposite provides a large surface for aptamer loading. The RGO‐Au/aptamer was combined with Nafion polymer and immobilized on a glassy carbon electrode. The interaction of aptamer with PSA was studied by cyclic voltammetry (CV), square wave voltammetry (SW), and electrochemical impedance spectroscopy (EIS). The detection of limit for prepared electrode was obtained about 50 Pg/ml at the potential of 0.4V in Potassium hexacyanoferrate [K4 Fe (CN)6] medium. To decrease the LOD and applied potential of the prepared nanoprobe CuCQD is introduced as a new redox probe. The results show that this new electrochemical medium provides better conditions for the detection of PSA. LOD of nanoprobe in CuCQD media was obtained as 40 pg/ml at the potential of ‐0.2V. Under optimal conditions, the aptasensor exhibits a linear response to PSA with a LOD as small as 3 Pg/ml. The present aptasensor is highly selective and sensitive and shows satisfactory stability and repeatability. This article is protected by copyright. All rights reserved

Nanocomposite Hydrogels: A Promising Approach for Developing Stimuli-responsive Platforms and their Application in Targeted Drug Delivery

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Sep 2021

Introduction: With the development of hydrogels from half a century ago, their application in various medical fields, including drug delivery has been widely expanded. Hydrogels used in this field are produced with synthetic polymers such as polyvinylpyrrolidone, polyvinyl alcohol, or natural polymers like chitosan, agarose, and hyaluronic acid to develop biocompatible, biodegradable, and non-immunogenic drug carriers. However, limitations such as inadequate response to stimulation, low homogeneity, and poor loading capacity for hydrophobic drugs have limited the use of hydrogels for drug delivery due to the hydrophilic nature of the hydrogel. The use of nanoparticles in the structure of hydrogels to produce hydrogel nanocomposites leads to more diverse interactions such as hydrogen and electrostatic bonds in addition to covalent interactions between hydrogel polymers. In addition to enhancing the mechanical properties of the hydrogel and further homogeneity, these interactions lead to the formation of platforms responsive to various stimuli, attaining sustained release, and ameliorating the poor loading of hydrophobic drugs used in cancer treatment and wound dressing. Conclusion: A review of a research conducted in the last 20 years represents that the application of nanocomposite hydrogels in drug delivery includes a wide range of production methods, nanoparticles to create various stimulation mechanisms, and therapeutic applications. Indeed, research has been focused on developing smart systems for controlled release with stimuli to reduce side effects of conventional cancer treatment methods, such as chemotherapy, by targeting drug delivery and reducing drug administration frequency and mitigating chronic wound complications by the release of growth factors.

Beta-carotene/cyclodextrin-based inclusion complex: improved loading, solubility, stability, and cytotoxicity

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J Incl Phenom Macrocycl Chem

Beta-carotene (BC) is a vitamin A precursor and has potential anticancer benefits, but the delivery of BC is hindered by its low solubility and storage instability. To overcome these challenges, this study investigated the use of fabricated cyclodextrin-based nanosponges (CDNS) using different ratios of two cross-linkers, epiclon (EPI) and hexamethylene diisocyanate (HMDI) to form inclusion complex with BC. The ratios of crosslinkers to βCD for two most optimaly encapsulated CDNSs-BC were determined to be 2:1 for EPI and 4:1 for HMDI with loading efficiency of 61.46% and 59.61%, respectively. The charachterization tests were carefully done for two optimal CDNSs. Encapsulation significantly improved the solubility by ~ 10 folds, 30-day storage stability by 40% compared to BCs. The in vitro release of the two encapsulated products showed no burst release. The MTT assay revealed a variable increase in cytotoxic effect in both normal and cancer cells compared to free BC. Overall, the CDNSs appear to be promising carriers for the delivery of BCs.

Apoptosis Induction, Cell Cycle Arrest and Anti-Cancer Potential of Tamoxifen-Curcumin Loaded Niosomes Against MCF-7 Cancer Cells

Article

Apr 2022

Background & Objective: Breast cancer is the most common cancer among women. One of the most effective treatments for breast cancer is chemotherapy, in which specific drugs destroy the mass and its proliferation is inhibited. Chemotherapy is the most effective adjunctive therapy when multiple medications are used concurrently. Also, combining the drugs with nanocarrier has become an important strategy in targeted therapy. This study is designed to assess the apoptosis induction, cell cycle arrest, and anti-cancer potential of Tamoxifen-Curcumin-loaded niosomes against MCF-7 Cancer Cells. Methods: A novel niosomal formulation of tamoxifen-curcumin with Span 80 and lipid to drug ratio of 20 was employed. The MCF-7 cells were cultured and then treated with IC50 value of tamoxifen-curcumin-loaded niosomes, the combination of tamoxifen and curcumin, tamoxifen, and curcumin alone. Flow cytometry, Real-Time PCR, and cell cycle analysis tests were conducted to evaluate the induction of apoptosis. Results: Drug-loaded niosomes caused up-regulation of bax and p53 genes and down-regulation of bcl2 gene. Flow cytometry studies showed that niosomes containing tamoxifen-curcumin increased apoptosis rate in MCF-7 cells compared to the combination of tamoxifen and curcumin owing to the synergistic effect between the two drugs along with higher cell uptake by formulation niosomal. These results were also confirmed by cell cycle analysis. Conclusion: Co-delivery of curcumin and tamoxifen using optimized niosomal formulation revealed that at acidic pH of MCF-7 cancer cells, released drugs from niosomal carriers would be more effective than physiological pH. This feature of niosomal nanoparticles can reduce the side effects of drugs in normal cells. Niosomal nanoparticles might be used as a biological anti-cancer factor in treatment of breast cancer.

Engineering tumor stromal mechanics for improved T cell therapy

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Jan 2022
BBA-GEN SUBJECTS

Adoptive cellular therapies (ACT), including the engineered T cell receptor (TCR) therapy and chimeric antigen receptor (CAR) T Cell Therapy, are currently at the forefront of cancer immunotherapy. However, their efficacy for the treatment of solid tumors has not been confirmed. The fibrotic stroma surrounding the solid tumor has been suggested as a main barrier in the disarmament and suppression of the engineered T cells. In this review, we will discuss the recent findings on the mechanism of T cell suppression by the tumor stroma with a special emphasis on the effect of stromal mechanics. We will also discuss the engineering approaches used to dissect the mechanism of the T cell suppression by the stromal mechanical factors. Finally, we will provide a future outlook on the strategies to improve the efficacy of T cell therapy through altering the tumor stromal fibrosis.

The synthesis and characterization of double nanoemulsion for targeted Co-Delivery of 5-fluorouracil and curcumin using pH-sensitive agarose/chitosan nanocarrier

Article

Sep 2021
J DRUG DELIV SCI TEC

Due to the dangerous side effects of chemotherapy, advanced methods for curing cancer, such as drug delivery, have attracted much attention because of their high efficacies. Using a green synthesizing method, we prepared a double nano-emulsion agarose/chitosan nanocomposite as the carrier of this work to adjust stability and hydrogel releasing. Curcumin, 5-fluorouracil, and curcumin/5-fluorouracil were loaded into the nano-carrier, and the synthesized systems were characterized by XRD, FESEM, FTIR, DLS, and zeta potential analysis. Loading efficacy and entrapment efficacy of curcumin in the agarose/chitosan nanocomposite were 85.6% and 79.2%, respectively. Drug release profiles proved the pH sensitivity of the nanocomposite by providing a 24–44% difference in drug cumulative release rate between the tumor-simulated and the normal cell-simulated buffers. Also, MTT and the flow cytometry analysis showed the biocompatibility of the carrier and the effect of using both drugs, as a co-delivery system, on cytotoxicity against the MCF-7 cells. These results demonstrate the potential of the chitosan/[email protected]/5-fluorouracil to be a candidate for the targeted treatment of cancer cells.

Ultra pH-sensitive nanocarrier based on Fe2O3/chitosan/montmorillonite for quercetin delivery

Article

Sep 2021
INT J BIOL MACROMOL

Harmful side effects of the chemotherapeutic agent have been investigated in many recent studies. Since Fe2O3 nanoparticles have proper porosity, they are capable for loading noticeable amount of drugs and controlled release. We developed Fe2O3/chitosan/montmorillonite nanocomposite. Quercetin (QC) nanoparticles, which have fewer side effects than chemical anti-tumor drugs, were encapsulated in the synthesized nanocarrier and were characterized by X-ray diffraction (XRD), Fourier transforms infrared spectroscopy (FTIR), field emission scanning electron microscopy (FE-SEM), vibrating sample magnetometer (VSM), dynamic light scattering (DLS), and zeta potential. For quercetin, the encapsulation efficiency and the loading efficiency of the drug in Fe2O3[email protected] were found to be about 94% and 57%, respectively. The release profile of QC in different mediums indicated pH-dependency and controlled release of the nanocomposite, adhering to The Weibull kinetic model. Biocompatibility of the Fe2O3/CS/MMT nanoparticles against the MCF-7 cells was shown by MTT assay and confirmed by flow cytometry. These data demonstrate that the designed Fe2O3[email protected] would have potential drug delivery to treat cancer cells.

A novel pH‐responsive nanoniosomal emulsion for sustained release of curcumin from a chitosan‐based nanocarrier: Emphasis on the concurrent improvement of loading, sustained release, and apoptosis induction

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