Review: Preparation of Flavonoid Nanoparticles using the Nanoprecipitation Method

Rizky Farhan Pratama; Iyan Sopyan; Taofik Rusdiana

Vol 4, Issue 4, 2023 (307-332)

http://journal.unpad.ac.id/idjp

*Corresponding author,

e-mail : rizky17009@mail.unpad.ac.id (R. F. Pratama)

https://doi.org/10.24198/idjp.v4i3.40849

Review: Preparation of Flavonoid Nanoparticles using the Nanoprecipitation Method

Rizky Farhan Pratama*,1, Iyan Sopyan2, Taofik Rusdiana2

1Bachelor Program in Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran

2Department of Pharmaceutical and Formulation Technology, Faculty of Pharmacy,

Universitas Padjadjaran Jalan Raya Bandung-Sumedang KM 21, Jatinangor

45363, Indonesia

Submitted : 22/07/ 2022, Revised : 01/08/ 2022, Accepted : 06/06/2023, Published : 16/08/2023

Abstract

Flavonoids are polyphenolic compounds that have 15 carbon chains, 2 benzene

rings and a heterocyclic pyran ring. From the literature study, it is known that

flavonoids have various pharmacological activities such as anticancer,

antimicrobial, antiviral, antiangiogenic, antimalarial, antioxidant, neuroprotective,

antitumor, and antiproliferative agents. However, flavonoids have limited oral

bioavailability which may be due to their poor solubility, low permeability, and low

stability, which impair their effectiveness as therapeutic agents. One of the efforts

to increase solubility is nanoparticle technology where the active compound

particles are reduced to the nanometer scale, usually up to 100 nm.

Nanoprecipitation is a method of preparing nanoparticles by dissolving the active

drug substance and polymer into an organic solvent and then adding an anti-solvent

such as water. The advantages of this method are the production is relatively fast,

inexpensive, does not require a lot of energy, and does not require emulsion

precursors. The purpose of this literature review is to examine the technique of

making flavonoid nanoparticles using the nanoprecipitation method, the results of

their characterization and evaluation. Based on a literature review that has been

carried out on 30 journals, there are 20 flavonoid secondary metabolites that have

been prepared into nanoparticles using the nanoprecipitation method. Some of the

polymers used were effective in achieving satisfactory particle size, polydispersity

index (PDI), Zeta potential and Encapsulation Efficiency (EE%). Thus, the

nanoprecipitation method can be used to make flavonoid nanoparticles with optimal

formulations to improve the physicochemical properties of flavonoids for drug

development in the future.

Keywords: Flavonoid, Nanoparticles, Nanoprecipitation Method, Characterizatio

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308

1. Introduction

Flavonoids are polyphenolic

compounds that have 15 carbon chains, 2

benzene rings and a heterocyclic pyran ring.

Based on existing literature studies,

flavonoids have various pharmacological

activities such as anticancer, antimicrobial,

antiviral, antiangiogenic, antimalarial,

antioxidant, neuroprotective, antitumor, and

antiproliferative agents. One example of a

compound that has pharmacological activity

is quercetin which has anticancer activity.

However, some flavonoid compounds,

especially flavonoids which are included in

the category of flavonoid aglycones have low

solubility, low absorption and short drug

residence time [1,2].

In order to overcome these problems,

physical and chemical modifications of drugs

and various methods were carried out. The

methods used were about reducing particle

size, formation of co-crystal, formation of

salt, solid dispersion, surfactants appliaction,

complexation methods, approaches with

nanotechnology and so on [3,4]. Several

studies have approached nanotechnology to

make stable formulas. Nano technology is

used to reduce the particle size of a particle

so that it will form nanoparticles.

Nanoparticles are substances that have a

diameter of less than 1000 nm [5]. Utilization

of nano technology or the manufacture of

nanoparticles can be used for flavonoid

modification. One example of the research

conducted by Telange et al. which proves that

the solubility of Apigenin can be increased by

the solubility value of Apigenin-phopolipid

Phytosome in water by 22.80 g/mL. The

formation of nanoparticles also serves to

increase anticancer activity, for example, the

formation of quercetin nanoparticles with

PLGA polymer which prevents the formation

of hepatocellular carcinoma through the

protection of the mitochondrial membrane in

the liver [6]. The simplest method of forming

nanoparticles is Nanoprecipitation.

Nanoprecipitation is a method of

preparing nanoparticles for hydrophobic

active substances developed by Fessi et al in

1989. The principle behind this method is

using the Marangoni effect. Overall this

method is carried out by making the active

drug substance and polymer dissolved in an

organic solvent which is then added to an

anti-solvent such as water. The advantages of

this method are that the production is

relatively fast, low cost, does not require a lot

of energy, and does not require emulsion

precursors. In addition, nanoprecipitation can

also produce nanoparticles with particle sizes

in the range of 50-300 nm [5,7,8,9].

Based on the literature search, the

preparation of nanoparticles with the

nanoprecipitation method has been widely

carried out on natural materials including

flavonoids. Therefore, a literature review was

carried out by collecting data on the

manufacture of flavonoid nanoparticles with

the nanoprecipitation method as a reference

for future research on the manufacture of

flavonoid nanoparticles.

2. Methods

This review was carried out by

conducting a literature search related to the

manufacture of flavonoid nanoparticles using

the nanoprecipitation method and their

characterization on the ScienceDirect,

Pubmed and Springer Link sites with specific

keywords used to search for qualified articles

such as "Flavonoid", "Nanoparticle",

"Nanoprecipitation". Method”, and

“Characterization”. The articles used are

articles from the last 10 years in English.

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309

Figure 1. Methods performed for review

3. Results and Discussion

Table 1. Variety of the Nanoprecipitation method used in the preparation of Flavonoid

Flavonoid

Nanoparticle

Technologies

Nanoparticl

e Types

Nanoprecipitatio

n method

Carrier

Solubility/Bioavailability

/Dissolution

Reference

Apigenin

Apigenin-

Polimeric

Traditional

PLGA

[27]

Search articles using keywords: “Flavonoid”, “Nanoparticle”, “Nanoprecipitation

Method”, and “Characterization” on ScienceDirect, Pubmed and Springer Link.

Initial search results (n = 301)

Reprocessed article search results

(n = 118)

Unprocessed article from search

results (n = 183)

Articles used in research (n = 30)

Exclusion critetia:

•

he article does not discuss the

manufacture of flavonoid

nanoparticles using the

nanoprecipitation method and

their characterization

•Articles are not research

articles (such as reviews and

systematic reviews.)

•Articles are not in English

language

Inclusion critetia:

•

he article discuss the

manufacture of flavonoid

nanoparticles using the

nanoprecipitation method and

their characterization

•

Articles are research articles

•Articles published from the last

10 years

•Articles are in English

language

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310

loaded PLGA

nanoparticles

Nanoparticl

Nanoprecipitatio

Apigenin-

loaded

galactose

tailored

PLGA

nanoparticles

Polimeric

Nanoparticl

Traditional

Nanoprecipitatio

Galaktosa dan

PLGA

[27]

Artocarpin

Nanoparticle

system

(Artocarpin:

PVP)

Polimeric

Nanoparticl

Heat induced

evaporative

antisolvent

nanoprecipitatio

PVP

Solubility: 1400-fold

[28]

Chrysin

nanocapsules

Lipid-

polymer

hybrid

Nanoparticl

Traditional

Nanoprecipitatio

PLGA,

Labrafac PG,

phosphatidylch

oline

[29]

Cirsiliol

Cirsiliol-

loaded

nanocapsules

Lipid-

polymer

hybrid

Nanoparticl

Traditional

Nanoprecipitatio

PEG-PCL, Span

80, Tween 80

Solubility: 24-fold

[30]

Curcumin

encapsulated

Chitosan

functionalized

PLGA Core

Shell

Nanoparticles

Organic-

Inorganic

hybrid

Nanoparticl

Traditional

Nanoprecipitatio

PLGA, Kitosan

[31]

PLGA-CTAB

curcumin

nanoparticles

Polimeric

Nanoparticl

Heat induced

evaporative

antisolvent

nanoprecipitatio

PLGA, CTAB

[32]

Dihydromyricet

Nanocapsule

suspensions

containing

Dihydromyric

etin

Polimeric

Nanoparticl

Traditional

Nanoprecipitatio

Eudragit RS100

[33]

Diosmin

Polymer-

stabilized

diosmin

nanosuspensi

Polimeric

Nanoparticl

Traditional

Nanoprecipitatio

HPMC/MC

Dissolution: 2-fold

[34]

Eupafolin

nanoparticles

Polimeric

Nanoparticl

Traditional

Nanoprecipitatio

PVA, Eudragit

E100

[35]

Fisetin

Fisetin-

loaded

nanoparticles

Polimeric

Nanoparticl

Traditional

Nanoprecipitatio

PCL, PLGA-

PEG-COOH,

Pluronic F127.

[12]

Genistein

Genistein-

loaded M-

PLGA-TPGS

Polimeric

Nanoparticl

Traditional

Nanoprecipitatio

M-PLGA-

TPGS

[26]

Luteolin

Hybrid

Polimeric

Heat induced

PLA, Eudragit

Bioavaialbility: 2.61-fold

[36]

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311

PLA/Eudragit

100 Luteolin

nanoparticles

Nanoparticl

evaporative

antisolvent

nanoprecipitatio

L100, Pluronic

F127

Naringenin

PVP-coated

naringenin

nanoparticles

Polimeric

Nanoparticl

Sonication-

assisted

nanoprecipitatio

PVP

[37]

Naringenin-

loaded

nanoparticles

Polimeric

Nanoparticl

Traditional

Nanoprecipitatio

Eudragit, PVA

[25]

Phloretin

Hydrogel

containing

polymeric

nanocapsules

loaded

with phloretin

Polimeric

Nanoparticl

Heat induced

evaporative

antisolvent

nanoprecipitatio

PCL, Span 60,

Tween 80,

[38]

Poly-puerarin

nanoparticles

with

Paclitaxel

Polimeric

Nanoparticl

Traditional

Nanoprecipitatio

Pluronic F127

[39]

Proantosianidin

PLGA

nanoparticles

loaded with

Proamthocian

idin-rich

Grapeseed

Extract,

Polimeric

Nanoparticl

Traditional

Nanoprecipitatio

PLGA

[40]

Quercetin

P. guajava

ethyl acetate

fraction

loaded

nanosuspensi

Polimeric

nanoparticle

Sonication-

assisted

nanoprecipitatio

PVA

Bioavailability: 6,28-fold

[15]

Starch

nanoparticles

loaded with

Quercetin

Starch

nanoparticle

Traditional

Nanoprecipitatio

Pati Kentang

[24]

MPEG-PLA

encapsulated

Quercetin

nanoparticle

Polimeric

nanoparticle

Traditional

Nanoprecipitatio

MPEG-PLA,

Pluronic F-68

Bioavaialability: 1.87-

fold

[16]

Quercetin

embedded

PLA

nanoparticles

Polimeric

nanoparticle

Sonication-

assisted

nanoprecipitatio

PLA, PVA

[41]

Zinc

phthalocyanin

e-Quercetin

loaded lipid-

polymer

hybrid

nanoparticle

Lipid-

polymer

hybrid

Nanoparticl

Heat induced

evaporative

antisolvent

nanoprecipitatio

PLGA, soybean

lecithin, DSPE-

PEG2000

Bioavailability: 3.64-fold

[42]

Quercetin-

loaded

Eudragit®

Polimeric

nanoparticle

Traditional

Nanoprecipitatio

Eudragit S100

[43]

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312

S100

Nanoparticles

Quercetin-

loaded PCL

based

nanoparticles

Polimeric

nanoparticle

Heat induced

evaporative

antisolvent

nanoprecipitatio

PCL

[44]

Quercetin

conjugated

Fe3O4

nanoparticles

Magnetic

nanoparticle

Sonication-

assisted

nanoprecipitatio

Dextran, Fe3O4

[11]

Rutin

nanospheres

Polimeric

Nanoparticl

Sonication-

assisted

nanoprecipitatio

Eudragit S100,

Poloxamer-188.

[45]

Salvigenin

Salvigenin-

loaded

mPEG-b-

PLGA with

Fe3O4

Magnetic

nanoparticle

Heat induced

evaporative

antisolvent

nanoprecipitatio

mPEG-b-

PLGA, Fe3O4

[23]

Silibinin

Silibinin-

loaded

nanoparticles

Polimeric

Nanoparticl

Traditional

Nanoprecipitatio

PVA, Eudragit

E100

[46]

Silymarin

Silymarin-

loaded lipid

polymer

hybrid

nanoparticles

containing

chitosan

Lipid-

polymer

hybrid

Nanoparticl

Heat induced

evaporative

antisolvent

nanoprecipitatio

PLGA, soybean

lecithin, DSPE-

PEG2000,

Kitosan

[47]

Silymarin-

Loaded

Eudragit

Polimeric

Nanoparticl

Sonication-

assisted

nanoprecipitatio

PVA, Eudragit

RS100 &

RL100

[14]

Table 2. Characterization of Flavonoid Nanoparticles Using Nanoprecipitation Method

Flavonoid

Nanoparticle

Technologies

Partikel

Size

(nm)

PDI

Zeta

Potential

(Mv)

EE (%)

Activity

References

Apigenin

Apigenin-loaded PLGA

nanoparticles

110,0

0,041 +

0,004

-25,0

70,3

Hepatocellular

Carcinoma Treatment

[27]

Apigenin-loaded

galactose tailored

PLGA nanoparticles

129,0

0,059 ±

0,007

-14,0

75,4

Hepatocellular

Carcinoma Treatment

[27]

Artocarpin

Nanoparticle system

(Artocarpin: PVP)

128,4 ±

0,7

0,266 ±

0.024

>99

Hepatocellular

Carcinoma Treatment

[28]

Chrysin

Chrysin nanocapsules

176 ±

2,10

0,22 ±

0,01

-6,23 ±

0,18

87,10 ±

6,71

Antiglycemic,

Antihyperlipidemic

[29]

Cirsiliol

Cirsiliol-loaded

nanocapsules

158.1 ±

12,4

0,19 ±

0,01

−2,6 ± 5.1

53,5 ±

2,1

Anticancer

[30]

Curcumin

Curcumin encapsulated

Chitosan functionalized

207,6 ±

2,71

0,165 ±

0,075

+31,9 ±

1,03

75.53 ±

2,09

Alzheimer’s Disease

Treatment

[31]

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313

PLGA Core Shell

Nanoparticles

PLGA-CTAB curcumin

nanoparticles

81,05 ±

3.85

0,107

+31,8

69,1

Breast Cancer

Treatment

[32]

Dihydromyricetin

Nanocapsule

suspensions containing

Dihydromyricetin

160 ± 5,0

0,120 ±

0,05

+8,5 ± 1,5

Photoprotection

[33]

Diosmin

Polymer-stabilized

diosmin

nanosuspension

316 ±

5,55

0,41 ±

0,04

[34]

Eupafolin

Eupafolin nanoparticles

90,8

Acute Renal Injury

Treatment

[35]

Fisetin

Fisetin-loaded

nanoparticles

198,7 ±

6,0

0,158 ±

0,02

74,78 ±

1,9

Antioxidant

[12]

Genistein

Genistein-loaded M-

PLGA-TPGS

225,7 ±

2,5

0,169

-14,2 ±

0,7

97,66

Liver Cancer

Treatment

[26]

Luteolin

Hybrid PLA/Eudragit

100 Luteolin

nanoparticles

452,23 ±

22,4

0,92 mV ±

0,04

71,02 ±

14,6%

[36]

Naringenin

PVP-coated naringenin

nanoparticles

110

99,93

Antioxidant

[37]

Naringenin-loaded

nanoparticles

Anticancer

[25]

Phloretin

Hydrogel containing

polymeric nanocapsules

loaded

with phloretin

252 ±

12,01

1,68 ±

0,11

>-1

>99

Antitumoral

[38]

Poly-puerarin

nanoparticles with

Paclitaxel

70,26

0,15

−23,1

91,3

Anticancer

[39]

Proantosianidin

PLGA nanoparticles

loaded with

Proamthocianidin-rich

Grapeseed Extract,

132,5 ±

12,2

–26,7 ±

3,8

65,2 ±

6,1

Dentin Degradation

Resistance

[40]

Quercetin

P. guajava ethyl acetate

fraction loaded

nanosuspension

241,32 ±

1,25

0,224 ±

0,011

-22 ± 0,4

92,85 ±

2,23

Antihyperglycemic

[15]

Starch nanoparticles

loaded with Quercetin

91,2 –

154,5

0,276 –

0,41

Antioxidant

[24]

MPEG-PLA

encapsulated Quercetin

nanoparticle

155,3 ±

3,2

0,2 ±

0,05

−3,14

Breast Cancer

Treatment

[16]

Quercetin embedded

PLA

nanoparticles

46 ± 4

62±3

Anticancer

[41]

Zinc phthalocyanine-

Quercetin loaded lipid-

polymer hybrid

nanoparticle

174,8

0,331

−30 ± 10

10 ± 4

Photodynamic

Anticancer

[42]

Quercetin-loaded

Eudragit® S100

Nanoparticles

66,8 ±

2,3

-5,2 ± 2,4

41,8 ±

9,1

Colon Cancer

Treatment

[43]

Quercetin-loaded PCL

based nanoparticles

215,9 ±

2,9

0,094

-12,9 ±

0,35

66,32 ±

0,4

[44]

Quercetin conjugated

Fe3O4 nanoparticles

+6,14

81,6

Breast Cancer

Treatment

[11]

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314

Rutin

Rutin nanospheres

130,30 ±

35,29

0,29 ±

0,10

-22,90 ±

5,18

98,10 ±

0,50

Colon

Carcinoma Treatment

[45]

Salvigenin

Salvigenin-loaded

mPEG-b-PLGA with

Fe3O4

57 ± 2

0,168 ±

0,03

−33 ± 1,2

82 ± 1,6

Anticancer

[23]

Silibinin

Silibinin-loaded

nanoparticles

120

+4,6

79,0 ±

2,4

Oral Carcinoma

Treament

[46]

Silymarin

Silymarin-loaded

lipid polymer hybrid

nanoparticles

containing chitosan

286,5 ±

23,8

0,226 ±

0,008

45,3 ± 8,9

97,05 ±

0,01

Hepatoprotective

Agent

[47]

Silymarin-Loaded

Eudragit

84,70

0,38 ±

0,01

83,45

Hepatoprotective

Agent

[14]

The formation of flavonoid

nanoparticles increases the oral

bioavailability of insoluble active flavonoid

components via particle reduction which

resulting in increase of surface are.

Nanoparticles formation also led to the

increase of saturation solubility and

dissolution rate. [10,11,12,13].

Particle size and polydispersity

index (PDI) influence the physicochemical

properties of nanoparticles such as

dissolution speed, saturation solubility, and

physical stability. Polydispertity index also

describes the uniformity of particle shape.

The effective nanosuspension size for

absorption in the intestine ranged from 100 to

500 nm. The nanosuspension size is in the

range of 100 - 200 nm. The ideal particle size

for in-vivo treatment is less than 200 nm.

Nanoparticles are said to be homogeneous if

their polydispersity index is less than 0.4.

PDI values greater than 0.4 indicate particle

aggregation [14,15,16,17,18].

Zeta potential is related to the

electrostatic potential that develops at an

indistinct boundary between the

nanoparticles and the solution. Nanoparticles

have is stable and have positive or negative

charge if the zeta potential value is greater

than +30 mV or less than -30 mV for positive

charge and negative charge respectively

[15,19,20].

Encapsulation Efficiency (EE%) is

the percentage of drug trapped into the

nanocarrier matrix which refers to the total

drug input. A good Efficiency of

Encapsulation has a value of >80%

[21,22,23].

Preparation of nanoparticles with the

Nanoprecipitation method is carried out by

adding a dilute polymer solution to a non-

solvent or vice versa which is then followed

by polymer deposition at the nanoscale

[14,24,25,26].

The traditional Nanoprecipitation

method is prepared in large volumes of

solution by adding an anti-solvent to a

solvent containing hydrophobic molecules or

vice versa drop by drop under mixing. In

small-volume mixing processes, this process

produces particles instantaneously with a

narrow size distribution often at the

nanoscale. Anti-solvents must be miscible

with solvents containing polymers or drugs.

Solutions with large volumes are difficult to

control the results with the stirring process

[48]. The Heat induced evaporative

antisolvent nanoprecipitation (HIEAN)

method begins by dissolving the active

R F. Pratama et al / Indo J Pharm 4 (2023) 307-332

315

substance in an organic solvent at a low

boiling point. Then, the resulting solution is

added to the heated aqueous solution. The

increased temperature of the heated aqueous

solution ensures rapid evaporation of organic

solvents and results in a high degree of

saturation and rapid precipitation of the drug

in the form of suspended particles. Stabilizers

added to a heated aqueous solution adsorb

onto the surface of newly formed particles

and reduce the surface energy and prevent

particle growth [49, 50].

The nanoprecipitation method

approach with the help of sonication is

applied to reduce the particle size of the

active substance which is not soluble in

water. Ultrasonication into the carrier

containing solution is carried out to coat and

stabilize the nanoparticles. The propagation

of ultrasound into a liquid medium results in

alternating cycles of compression and

contraction resulting in vacuum bubbles

which accumulate energy and then release it

violently or it is also called cavitation.

Cavitation can trigger and accelerate various

chemical reactions including the formation of

nanoparticles [51].

The nanoparticles that are often

made by this method are polymeric

nanoparticles. Polymeric nanoparticles have

more stability in the gastrointestinal tract than

other colloidal carriers. Polymeric

nanoparticles have other advantages such as

maintaining effect on target tissues, solubility

for intravascular delivery and protection from

enzymatic degradation, especially in gastric

acid. The polymers that are often used are

polymers from the polyester family such as

PLGA, PLA and PCL [14,24,25,26].

Based on the results of the literature

research that has been carried out, 301

articles were obtained which were then

processed into 30 articles. The article

research process was carried out using

indexed databases such as ScienceDirect (n =

24), PubMed (n = 4) and SpringerLink (n =

2). The results of literature research can be

found in Table 1.

3.1 Apigenin

Apigenin was prepared into two

different nanoparticles, the first nanoparticle

using PLGA as a carrier while the second

nanoparticle using Galactose-PLGA as a

carrier. Both nanoparticles use the same

method. The manufacture of nanoparticles

starts from dissolving the polymer (PLGA or

Galactose-PLGA) and Apigenin in acetone.

The mixtures were poured into ultrapure

water containing poloxamer 188 [27].

Nanoparticles with PLGA produced

have particle size of 110 nm, polydispersity

index 0.041 ± 0.004, Zeta Potential -25.0 mV

and Efficiency of 70.3%. Nanoparticles with

Galactose-PLGA produced a particle size of

129.0 nm, polydispersity index 0.059 ±

0.007, Zeta Potential -14.0 mV and

encapsulation efficiency of 75.4%. The low

polydispersity index value indicates that the

nanoparticles show the same size between

each other. The zeta potential value of

nanoparticles made with Galactose-PLGA is

smaller than using only PLGA. This is due to

the PLGA galactosylation. The negative zeta

potential also aids the uptake of the

reticuloendothelial system (RES) [27].

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316

3.2 Artocarpin

Artocarpine which has an autophagic

effect was made into naooparticles with an

artocarpine: PVP nanoparticle system with a

ratio of 1:10. The steps start from dissolving

artocarpine in ethanol to form organic phase.

The aqueous phase is prepared by adding

PVP in ultrapure water. The organic phase

was then injected into the aqueous phase. The

resulting nanoparticles had a particle size of

128.4 ± 0.7 nm, a polydispersity index of

0.266 ± 0.024, an encapsulation efficiency

above 99% and a water solubility of 1400

times that of free Artocarpin. The particle

size below 200 nm makes administration of

the drug in the body more effective. The PDI

value indicates the absence of agglomerates.

Formation of intermolecular hydrogen bonds

between the OH bonds of artocarpin and the

CO bonds of PVP increase solubility [28].

3.3 Chrysin

Chrysin nanocapsules were made

using Labrafac PG as oil, PLGA as polymer

and Phosphatidylcholine (PC) and Tween 80

as surfactants. The resulting nanocapsules

had a particle size of 176 ± 2.10 nm, a

polydispersity index of 0.22 ± 0.01, a Zeta

Potential of -6.23 ± 0.18 mV, and an

encapsulation efficiency of 87.10 ± 6.71%.

The obtained small particle size nanocapsules

provide a high mucosal adhesion ability on

the gastrointestinal surface, which ensures a

longer retention time. The PDI values of the

nanocapsules showed a good size

distribution. The increase of polymers and

surfactants caused an increase in the particle

size of the nanocapsules. This is due to an

increase in the viscosity of the polymer

solution, making it difficult for the

emulsification process to become smaller

droplets. Increasing the concentration of

tween 80 decrease particle size at a constant

amount of polymer and drug. This is due to a

reduction in the interfacial tension between

dispersed organic phase and dispersion

medium. The zeta potential value obtained is

too low but tween 80 which is reported to

stabilize nanoparticles with a steric effect will

provide protect nanocapsules stability even

though their zeta potential value is low [29].

3.4 Cirsiliol

Picture 1. Production of Cirsiliol nanocapsules [30]

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317

Nanocapsules containing Cirsiliol

were prepared using the modified

nanoprecipitation method by adding oil and

lipophilic surfactants to the organic phase and

adding hydrophilic surfactants to the aqueous

phase. The method produces colloidal balls

with an oil-filled core. The organic phase

consisted of castor oil, cirsiliol, PEG-PCL

and Span 80 which were dissolved in a

mixture of acetone and ethanol while the

aqueous phase consisted of Tween 80

dissolved in water. PEG is used as a carrier

because PEG has a slow degradation due to

its high crystallinity level, thereby prolonging

drug release and maintaining drug stability.

To improve PEG performance, PCL and PEG

chains were combined by polymerizing CL

ring opening using mPEG as a macroinitiator

and stannous octoate as a catalyst. The

resulting nanocapsules have a particle size

characteristic of 158.1 ± 12.4 nm, a

polydispersity index of 0.19 ± 0.01, a Zeta

Potential of 2.6 ± 5.1 mV, an encapsulation

efficiency of 53.5 ± 2.1% and an increase in

solubility of 24 times that of ordinary

Cirsiliol [30].

3.5 Curcumin

1. Curcumin encapsulated Chitosan

functionalized PLGA Core Shell

Nanoparticles

Core/Shell nanoparticles are

suitable for drugs that target the brain. In

addition, this type of nanoparticles can

also protect the drug from P-gp efflux and

external degradation [31].

In this study, used the organic

phase consisting of curcumin and PLGA

dissolved in acetone and the aqueous

phase consisting of pluronic F127

dissolved in water. PLGA used as core

material because it is biocompatible,

biodegradable and non-toxic. This

polymer can also be used in conjunction

with other polymeric materials. After the

nanoparticle solution is formed, chitosan

is added to the solution so that

electrostatic interactions occur which will

produce Curcumin encapsulated Chitosan

functionalized PLGA Core Shell

Nanoparticles. Chitosan is able to

increase the residence time of intranasal

drugs. This is because the positive charge

interacts electrostatically with the mucin.

Chitosan also has the ability to form a gel

by absorbing water [31].

The characterization results

showed that the nanoparticles made had a

particle size of 207.6 ± 2.71 nm, a

polydispersity index of 0.165 ± 0.075, a

Zeta Potential of +31.9 ± 1.03 mV, and an

encapsulation efficiency of 75.53 ±

2.09%. Particle size and polydispersity

index increased with increasing

concentration of curcumin in the

formulation. Particle size also increased

by coating chitosan against PLGA [31].

2. PLGA-CTAB curcumin

nanoparticles

Curcumin will be used as an

anticancer by making nanoparticles by

applying PLGA as carrier and

hexadecyltrimethylammonium bromide

(CTAB) as a surfactant. PLGA and

curcumin were added to the mixture of

Acetone: Ethanol (17:3) to make an

organic phase while CTAB was dissolved

in water to make an aqueous phase.

PLGA is a polymer with negatively

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charged molecules which is often used as

a carrier in the manufacture of

nanoparticles. However, positively

charged nanoparticles move more easily

from the endosome to the cytosol so that

their bioavailability is better than neutral

or negatively charged nanoparticles. To

produce positively charged nanoparticles,

CTAB which works as a surfactant as

well as a molecular charge modifier is

added during the nanoparticle

manufacturing process [32].

The characterization results

showed that the nanoparticles made had a

particle size of 81.05 ± 3.85 nm, a

polydispersity index of 0.107, a Zeta

Potential of +31.8 mV, and an

encapsulation efficiency of 69.1%. The

small particle size may be due to the

addition of CTAB. The results of the

Polydispersity Index stated that no

aggregates were formed. The zeta

potential obtained indicates that the

nanoparticles are stable enough so that

there is no aggregate formation. The

results of encapsulation efficiency are

influenced by the presence of PLGA

which has a low molecular weight [32].

3.6 Dihydromyricetin

Dihydromyristetin nanocapsules are

made by preparing a solution of Eudragit first

and then adding Dihydromyristetin. To make

hydrogel, 1.5% Hydroxyethylcellulose,

methylparaben and propylene glycol were

added. Hydroxyethylcellulose works as a

thickening agent. The resulting nanocapsules

had a particle size of 160 ± 5.0 nm, a

polydispersity index of 0.120 ± 0.05 and a

Zeta Potential of 8.5 ± 1.5 mV. The

nanoparticles made have a narrow particle

size distribution but their stability is still

questionable because the zeta potential value

is close to zero. Zeta Potential with a positive

charge is needed in order to provide

electrostatic attraction with a negative zeta

potential on human skin, causing an

occlusive effect on the skin [33].

3.7 Diosmin

Diosmin made nanoparticles with two

nanoprecipitation methods, acid-base

neutralization method and anti-solvent

precipitation method. The polymers used as

stabilizers were

Hydroxypropylmethylcellulose (HPMC) and

methylcellulose (MC) [34].

The step of the acid-base

neutralization method in a nutshell starts with

dissolving Diosmin in 1 N NaOH. Diosmin

base solution was dropped into 0.1 N HCl

containing polymer stabilizer, HPMC or MC.

For certain formulations, it is necessary to

remove excess stabilizer and then remove it

by ultracentrifugation. The supernatant was

discarded and then re-dispersed in water [34].

For the antisolvent precipitation method step,

Diosmin was dissolved in DMSO then added

to an aqueous solution containing 0.15%

polymer, HPMC or MC. Stirring was

maintained until a homogeneous milk

suspension was obtained [34].

The nanoparticles with the best

characterization results had an MC

concentration of 14% with the addition of

53% mannitol accompanied by spray-drying

and using an acid-base neutralization method.

The best nanoparticles had a particle size of

316 ± 5.55 nm and a polydispersity index of

0.41 ± 0.04. The anisolven method produces

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319

a higher PDI than the acid-base neutralization

method. The higher concentration of

stabilizer was reported not to significantly

affect the particle size, but the nanoparticle

formulation of the precipitation took longer

[34].

3.8 Eupafolin

In order to overcome the poor

solubility of Eupafolin, a research was

conducted on the manufacture of Eupafolin

nanoparticles. The reason why Eupafolin is

made into nanoparticles is because in

previous studies Eupafolin nanoparticles

have the potential to create new strategies in

dealing with acute kidney injury. In this

study, Eupafolin nanoparticles were prepared

using the nanoprecipitation method with

solvent evaporation. The organic phase

consisted of Eupafolin, Eudragit and 95%

alcohol while the aqueous phase consisted of

PVA and water. Eudragit E100 and PVA

were chosen as polymers because of their

non-toxic, water-soluble and often used for

oral treatment. The resulting nanoparticles

have a particle size of 90.8. Nanoparticles of

this size are effectively used for in-vivo

treatment [35].

3.9 Fisetin

In this study, fisetin nanoparticles

were made from an organic phase containing

Poly-(ε-caprolactone) (PCL), PLGA-PEG-

COOH block copolymer and fisetin in

acetonitrile and the aqueous phase only

consisted of water. Before fabricating

nanoparticles, Sechi et al. do the PLGA-PEG-

COOH conjugation first. PLGA polymer was

chosen to be a polymer because PLGA can

protect the active substance from

degradation, reduce side effects and form

sustained drug release types. To improve the

performance of PLGA, modification of the

PLGA surface was carried out with

poly(ethylene glycol) (PEG). PEG can

prevent the opsonins binding, prolongs

circulation time in blood, facilitate tissues

targeting and reduces PLGA’s absorption by

Rapid Reticuloendothelial System (RES).

PCL is suitable to be used as a PLGA partner,

this is because PLGA and PCL will form

hydrophobic core. In addition, PCL is

permeable and non-toxic [12].

The characterization results showed a

particle size of 198.7 ± 6.0 nm, a

polydispersity index of 0.158 ± 0.02, and an

encapsulation efficiency of 74.78 ± 1.9%.

The particle size of the PLGA-PEG-COOH

nanoparticles is larger than that of ordinary

PLGA, this is because of hydrophilic PEG

chains present in external aqueous phase. The

nanoparticles showed a narrow and unimodal

particle distribution based on the obtained

PDI values. PCL results in better

encapsulation due to its high affinity for

Fisetin. The affinity decreases with

increasing concentration of PLGA-PEG-

COOH which is more hydrophilic [12].

3.10 Genistein

Genistein nanoparticles are made

using PLGA, this is because PLGA is part of

the polyester family which has good

biocompatibility and biodegradability.

However, PLGA and other polyester families

has disadvantage. Liver and RES can easily

eliminate and absorbed PLGA. This can be

solved by the addition of d-a-tocopheryl

polyethylene glycol 1000 succinate (TPGS).

In order to obtain nanoparticles with higher

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drug content and higher drug entrapment

efficiency, modification of the molecular

shape of the nanoparticles was carried out

into stars. Mannitol was used as the core of

the star polymer molecule in this study

because it has good chemical stability and is

suitable for use in the formulation of

anticancer tablets. The modified polymer was

named Mannitol-core PLGA-TPGS (M-

PLGA-TPGS) [26].

The organic phase consisted of M-

PLGA-TPGS alongside Genistein dissolved

by acetone while the aqueous phase consisted

of water. The characterization results showed

particle size of 225.7 ± 2.5 nm, polydispersity

index of 0.169, Zeta Potential of -14.2 ± 0.7

mV and encapsulation efficiency of 97.66 %.

The size of the nanoparticles is in the size

range that facilitates drug accumulation in

tumor blood vessels increased of drug

permeation and retention. Star-shaped

copolymers reduce particle size. The PDI

value indicates a relatively narrow size

distribution. Zeta potential is negative which

caused by ionized carboxyl groups from the

PLA and PGA. The encapsulation efficiency

is influenced by the binding affinity PLGA

which has star-shaped form and hydrophobic

Genistein [26].

3.11 Luteolin

In order to make biodegradable

luteolin nanoparticles, a biodegradable

polymer was chosen, polylactic acid (PLA).

Eudragit L100 is used in conjunction with

PLA as a polymer. This is because Eudragit

L100 is a pH-dependent polymer that is

resistant to gastric acidity and is soluble in

intestinal fluids. This polymer is suitable for

the active substance delivered to the large

intestine. In this research, two kinds of

organic phases were made. The first organic

phase has the composition Luteolin and

Eudragit L100 in Ethyl Alcohol. The second

organic phase is PLA in Dichloromethane.

The aqueous phase is Pluronic F127 which

acts as an emulsifier and is dissolved in HCl

pH 4 [36].

The resulting nanoparticles have a

particle size of 452.23 ± 22.4 nm, Zeta

Potential of 0.92 ± 0.04 mV, and

encapsulation efficiency of 71.02 ± 14.6%.

The higher concentration of total polymer

(combination of PLA and EUD100) led to the

production of larger nanoparticles. This is

due to an increase in the viscous force which

opposes the breakdown of the particles.

However, the viscous force increases

encapsulation efficiency. The presence of

PLA should make the zeta potential

negatively charged. The formation of a

PF127 layer on the particle surface makes the

surface charge a less negative value [36].

3.12 Naringenin

1. PVP-coated naringenin nanoparticles

Naringenin nanoparticles were

prepared by a simple nanoprecipitation

method using polyvinylpyrrolidone

(PVP) as a hydrophilic carrier. PVP is a

strong hydrophilic polymer which has

advantages such as delaying the

crystallization of compounds by forming

adduct molecules, non-toxic and

uncharged. PVP coating hydrophobic

surfaces increase biocompatibility and

reduce complement activation. The

manufacturing stage starts from

dissolving Naringenin in ethanol to make

the organic phase. The organic phase was

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rapidly injected into the aqueous solution

containing the PVP. The injection process

was carried out alongside sonication of

mixture. The particle size obtained is 110

nm with encapsulation efficiency of

99.93%. The nitrogen atom coordinated

with naringenin provides stability and

prevents agglomeration [37].

2. Naringenin-loaded nanoparticles

In another study, naringenin

nanoparticles were fabricated using

Eudragit E and PVA. Eudragit E and

PVA were used as polymers/carriers

simultaneously. Eudragit E was chosen

because Eudragit has the function of

increasing the solubility of drugs that are

poorly soluble in water and have a

dimethylamino group base site that is

ionized in gastric juice. Naringenin

nanoparticles were made by dissolving

Naringenin and Eudragit® E in ethanol to

make an internal organic phase. The

phase is rapidly injected into the external

aqueous phase containing PVA. The

particle size obtained is 90 nm.

Nanoparticles do not form agglomerates

or adhesions. Judging from the particle

size, these nanoparticles can cross the

vascular endothelium and accumulate at

the tumor site through the EPR effect

because the nanoparticle size is less than

400 nm [25].

3.13 Phloretin

Phloretin is made into Hydrogel

preparations containing Floretin-charged

polymer nanocapsules. The core shell of the

nanocapsule can resist degradation caused by

ultraviolet light. PCL is used as a polymer

because it has advantages such as excellent

drug loading capacity, controlled release rate

and slower degradation compared to

poly(glycolic acid) and other polymers [38].

Phloretin-charged polymer

nanocapsules were prepared by mixing an

organic phase consisting of copaiba oil,

sorbitan monostearate (Span 60), PCL and

Floretin in acetone:ethanol (8:1). The

hydrophilic phase contains polysorbate 80

(Tween 80) and water. Before making

hydrogel with the addition of Lecigel® 1%,

Nanocapsules were evaluated first. The

evaluation results showed a particle size of

252 ± 12.01 nm and a polydispersity index of

1.68 ± 0.11. The zeta potential is negatively

charged and close to zero, due to the presence

of polysorbate 80 at the particle/water

interface. The highest concentration of drug

that can be put into nanocapsules with

maximum encapsulation efficiency (>99%)

is phloretin 0.2 mg/Ml. The size distribution

is fairly narrow because of the polydispersity

index results obtained [38].

3.14 Poly-puerarin

Poly-puerarin nanoparticles with

Paclitaxel were prepared by using the

nanoprecipitation method. Poly-puerarin

serves as a carrier. Poly-puerarin is obtained

from the modification of Puerarin with

unsaturated olefins via acryloyl chloride

through free radical polymerization.

Azodiisobutyronitrile (AIBN) is used as an

initiator in the puerarin modification process.

Poly-puerarin nanoparticles were prepared by

nanoprecipitation and used as a carrier for

loading paclitaxel. Before making

nanoparticles, first Poly-puerarin was

dissolved in DMSO. The first oil phase was

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prepared by dissolving Paclitaxel in DMSO

at concentration. The second oil phase was

prepared by dissolving Stabilizer F-127 in a

different DMSO. The added buffer is saline

phosphate buffer [39].

In this study, there were two

variations of the formula where there was a

difference in the percentage of paclitaxel

used. Satisfactory results were obtained from

10% paclitaxel where the particle size

characterization results were 70.26 nm,

polydispersity index 0.15, Zeta Potential 23.1

mV and encapsulation efficiency of 91.3%.

Due to the intermolecular forces, increasing

the concentration of paclitaxel and puerarin

did not increase the particle size. The

hydrophobic drug Paclitaxel increases the

amount of the hydrophobic compound of the

nanoparticle complex which helps the

formation of a dehydrated and compact core.

Zeta potential is negatively charged due to

the strong electronegativity of glucose

groups, ester bonds and other functional

groups of poly-puerarin molecules [39].

3.15 Proantosianidin

Proanthocyanidins in grape seed

extract bind to type I collagen, inhibit matrix

metalloproteinase (MMP) activity and

decrease the rate of dentin demineralization.

To make a drug that can reduce the level of

demineralization of tink with gradual drug

release, Proanthocyanidin in the extract was

prepared in the form of nanoparticles with

different compositions of the ratio of PLGA

nanoparticles loaded with grape seed, which

have PLGA/Grape Seed Extract ratios of

100:25, 100:50, and 100:50. 75 w/w,

synthesized by a modified nanoprecipitation

technique [40].

The characterization results showed a

particle size of 132.5 ± 12.2 nm, Zeta

Potential –26.7 ± 3.8 mV and encapsulation

efficiency of 65.2 ± 6.1%. The nanoparticles

are uniformly spherical. The resulting

particle size aids drug delivery through the

exposed dentinal tubules of the

demineralized dentin. The OH functional

group present in the extract structure makes

the zeta potential negatively charged [40].

3.16 Quercetin

1. Psidium guajava ethyl acetate fraction

loaded nanosuspension

The ethyl acetate fraction from

Psidium guajava was made into nanoparticles

because this fraction has antihyperglycemic

activity. The fraction contains Quercetin

which has low solubility. The organic phase

was made by dissolving the ethyl acetate

fraction in ethanol while PVA was dissolved

in water. PVA works as a surfactant. The

characterization results showed that the

nanoparticles had a particle size of 241.32 ±

1.25 nm, a polydispersity index of 0.224 ±

0.011, a Zeta Potential of -22 ± 0.4 mV, and

an encapsulation efficiency of 92.85 ±

2.23%. The particle size is in the range of

100-500 nm so that absorption in the intestine

will be more effective. The polydispersity

index obtained shows the uniformity of

particle shape in the nanoparticles. The

optimal amount of PVA provides protective,

electrostatic stability to the particles and

reduces agglomeration. Zeta potential in the

range of 20-40 mV indicates the presence of

electrostatic stability [15].

2. Starch nanoparticles loaded with

Quercetin

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323

Quercetin nanoparticles are made by

utilizing starch made from corn, potatoes or

beans. Starch is a natural polymer that is

biodegradable, inexpensive and abundant.

However, starch has low solubility and is

sensitive to temperature and humidity. For

this reason, modifications are made with

nano technology. Starch nanoparticles

containing quercetin were prepared by

dissolving quercetin into a solution of

NaOH/urea/H2O (0.8 : 1: 98.2) containing

starch. After that, HCl was added.

NaOH/urea/H2O solution is used as a solvent

because it can dissolve cellulose. Urea has the

function of preventing the assemblage of

starch molecules [24].

The results showed that the suitable

starch to be used as a polymer was potato

starch. This is because corn starch and peanut

starch form aggregates after lyophilization,

while potato starch has a uniform nanofiber-

like structure. Potato starch nanoparticles

have a particle size of 91.2 – 154.5 nm and a

polydispersity index of 0.276 – 0.41 [24].

3. MPEG-PLA encapsulated Quercetin

nanoparticle

Methoxy poly(ethylene glycol)-

poly(lactide) (MPEG-PLA) polymer is used

as a carrier to make quercetin nanoparticles

which will be used as an agent to treat breast

cancer. The reason why a modified form of

PLA is used is because the nanoparticles with

the PLA polymer are rapidly cleared from the

circulatory system after systemic injection.

The copolymerization of PLA with

Polyethylene glycol (PEG) can improve these

limitations. In addition, the charge of the

nanoparticles will shift to a neutral charge,

the neutral charge helps the interaction of

polymer nanoparticles with the cell

membrane. MPEG provides a liquid

membrane on the outside of the nanoparticles

to resist hydrophobic drug release for drugs

that function as sustained-release doses [16].

The organic phase consisted of

MPEG-PLA and quercetin in acetonitrile.

The organic phase was added to a solution

containing pluronic F-68. The resulting

nanoparticles had a particle size of 155.3 ±

3.2 nm, a polydispersity index of 0.2 ± 0.05

and a Zeta Potential of 3.14 mV. Particle

sizes below 200 nm suggest that

nanoparticles can promote accumulation at

tumor sites. Polydispersity index below 0.4

indicates that the nanoparticles are in

homogeneous condition. The Zeta Potential

value obtained states that the nanoparticles

can avoid RES [16].

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4. Quercetin embedded PLA nanoparticles

Picture 2. Production of Quercetin embedded PLA nanoparticles [41]

Quercetin nanoparticles with

continuous release drug function were

prepared using the emulsion (o/w)

nanoprecipitation method. PLA is used as a

carrier while PVA is used as a stabilizer.

Quercetin and PLA allow the formulation of

sustained-release drugs. This is due to the

delayed diffusion and strong interaction

between Quercetin and PLA. In the

manufacture of nanoparticles, Quercetin is

dissolved in ethanol and then added PLA

which has been dissolved in

dichloromethane. After that, the mixture was

added with PVA dissolved in water [41].

In this study, a variant of the

formulation was made with the concentration

of PLA, PVA and changing temperature. The

best nanoparticles were produced from the

formulation with 10% Quercetin, 2% PVA

and 20 mg/ml PLA and the manufacturing

temperature was at 25oC. The nanoparticles

had a particle size of 46 ± 4 nm and an

encapsulation efficiency of 62 ± 3 %. The

size of the nanoparticles increased with

increasing PLA concentration. The particle

size gradually decreased with increasing

PVA concentration while the encapsulation

efficiency did not change drastically. The

lower particle size at higher temperatures is

due to their high mobility [41].

5. Zinc phthalocyanine-Quercetin

loaded lipid-polymer hybrid nanoparticle

Quercetin can function as photodynamic

therapy by making Lipid-polymer hybrid

nanoparticle (LPN) which encapsulates Zinc

phthalocyanine and Quercetin. Zinc-

phthalocyanine works as a second generation

photosensitizer while Quercetin as an

anticancer agent. Both substances must

accumulate in the tumor site. The LPN

structure consists of a non-aqueous polymer

core, a single or multiple lipid layer, and a

polyethylene glycol (PEG) conjugated lipid

layer. The polymer core encapsulates most of

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the Quercetin and Zinc-phthalocyanine. The

lipid layer facilitates absorption by cells. The

PEG-conjugated lipid layer increases the

circulation time of the active substance in the

body [42].

Picture 3. Production of Lipid-polymer hybrid nanoparticle [42]

LPN made a modified nanoprecipitation

method using PLGA as a carrier. In this

method PLGA diffusion occurs between two

different liquid phases. The organic phase

was prepared by dissolving Quercetin, Zinc-

phthalocyanine and PLGA in Acetone. The

aqueous phase was prepared by dissolving

PF-68, soy lecithin and DSPE-PEG in a

mixture of ethanol and water. The resulting

nanoparticles had a particle size of 155.3 ±

3.2 nm, polydispersity index 0.2 ± 0.05, Zeta

Potential 3.14 mV and encapsulation

efficiency of 10 ± 4 % for Quercetin and 55

± 5% for Zinc-phthalocyanine. Particle sizes

below 200 nm prove that the preparation is

effective for use by the intravenous route.

The concentration of Lecithin and Zinc-

phthalocyanine did not affect the particle size

while the increase in the concentration of

Quercetin and PLGA increased the particle

size. The presence of a single layer of DSPE-

PEG increased the stability of the preparation

[42].

6. Quercetin-loaded Eudragit® S100

Nanoparticles

Quercetin which will be used for colon

cancer drug manufacture of Quercetin non-

particles with Eudragit S100 polymer as a

carrier because other studies have proven an

increase in the bioavailability and stability of

the drug [43].

The material used consists of an organic

phase and an aqueous phase. The organic

phase consisted of Eudragit S100 and

Quercetin dissolved in ethanol. Eudragit

S100 is suitable for drugs targeted against the

colon. This is due to the carboxyl group of the

methacrylic acid moiety contained in

Eudragit S100. The carboxyl group will

ionize upon contact with neutral or alkaline

pH. This results in a negative repulsion

between the carboxylate side groups causing

the polymer to lose its charge. This carboxyl

group also makes the polymer insoluble in

the stomach [43].

The resulting nanoparticles are colloidal

dispersion like milk which is stable. This

nanoparticle has a particle size of 66.8 ± 2.3

nm, Zeta Potential -5.2 ± 2.4 mV and

Encapsulation Efficiency of 41.8 ± 9.1%. A

negatively charged zeta potential is generated

due to the presence of sodium lauryl sulfate

added by to Eudragit S100. The

encapsulation efficiency obtained indicates

that some of the quercetin may have been

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326

partitioned into the aqueous phase during the

nanoparticle formation process [43].

7. Quercetin-loaded PCL based

nanoparticles

Polycaprolactone (PCL) is used as a carrier

for the manufacture of quercetin

nanoparticles with various formulation

variants. PCL was chosen because of its high

permeability, biodegradability and

biocompatibility, non-toxic, non-mutagenic

and suitable for controlled release drugs. The

organic phase contains PCL and quercetin

dissolved in acetone. The aqueous phase

contains Pluronic F-127 as a stabilizer which

is dissolved in water. The resulting

nanoparticles have a particle size of 215.9 ±

2.9 nm, polydispersity index 0.094, Zeta

Potential 12.9 ± 0.35 mV and encapsulation

efficiency of 66.32 ± 0.4%. Increasing the

concentration of Pluronic F-127 increased the

particle size. Increasing the concentration of

PCL increases the viscosity of the organic

phase. The increase in viscosity increases the

diffusion resistance of the drug into the

aqueous phase and enhances the

encapsulation of the drug into nanoparticles

[44].

8. Quercetin conjugated Fe3O4

nanoparticles

The Nanoprecipitation method controls the

particle diameter and the unique magnetic

properties of the magnetite nanoparticles.

However, this method has limitations in the

manufacture of magnetic nanoparticles where

the agglomeration and distribution of

particles is not uniform, these limitations can

be overcome by the addition of urea. The

manufacturing process begins with

dissolving quercetin in acetone and

dissolving magnetite nanoparticles coated

with dextran in dimethyl sulfoxide (DMSO)

and deionized water. After that, the magnetite

nanoparticle solution was added with N-

hydroxy succinimide (NHS) and 1-ethyl-3-

(3-dimethylaminopropyl) carbodiimide

(EDC). Then the mixture was added to the

quercetin solution and the pH was adjusted

with KOH. Urea functions as an aggregation

controller and stabilizer to produce prism-

shaped particles. Dextran is a surfactant

which is used to control the shape of spherical

aggregates and to protect the phase change of

nanoparticles [11].

The best nanoparticles have a particle size of

72 nm, Zeta Potential 6.14 mV and

Efficiency of 81.6%. Particle size occurs due

to the conjugation of quercetin to Fe3O4

through the EDC/NHS reaction. The zeta

potential is positively charged due to the

strong electrostatic interaction between the

drug and the protonation of magnetite

through the carboxyl molecule and the

presence of anionic ions from the drug

molecule. Positive charge can increase drug

penetration into tumor cells [11].

3.17 Rutin

Rutin was made into nanospheres which were

prepared by the nanoprecipitation method.

Eudragit S100 functions as a polymer and

Poloxamer-188 as a stabilizer. Nanosphere is

a particle matrix in which the entire mass of

solid and drug particles is physically and

uniformly dispersed. Eudragit S100 was used

as a polymer because it does not degrade

below pH 7. In other words, this polymer is

insoluble in the pH of the stomach and

intestines, but soluble in the pH of the large

intestine (pH > 7) [45].

Nanosphere was prepared by dissolving

Eudragit S100 and rutin in a sealed bottle

containing methanol to make the organic

phase and dissolving Poloxamer-188 in

distilled water. The characterization results

show that the nanosphere has a particle size

of 130.30 ± 35.29 nm, polydispersity index

0.29 ± 0.10, Zeta Potential -22.90 ± 5.18 mV

and encapsulation efficiency of 98.10 ±

0.50%. The high encapsulation efficiency is

due to rutin has a high affinity for organic

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327

solvents that dissolve the dissolved polymer

and the interaction between rutin and

Eudragit S100 which indicates the formation

of intermolecular hydrogen bonds. The

polydispersity index value obtained indicates

that the nanoparticles are stable without any

agglomerates. The negative charge on the

zeta potential measurement was attributed to

the free acrylic acid group of Eudragit S100

as an anionic polymer [45].

3.18 Salvigenin

In order to overcome its poor bioavailability,

a study was conducted on the preparation of

Salvigenin loaded mPEG-b-PLGA coated

with iron oxide (Fe3O4) nanoparticles.

PLGA is used as a polymer because PLGA

has various intrinsic physical and chemical

properties such as monomer ratio (PL/GA),

drug loading potential, and controllable size

and shape of drug polymer particles. The

addition of iron oxide particles into the

polymer nanocarrier is very important

because it allows the nanoparticles to be

delivered by a magnetic field to the tumor site

[23].

Before making Salvigenin nanoparticles,

Fe3O4 nanoparticles must be made first with

the main ingredients FeCl3.6H2O and

FeCl2.4H2O with a molar ratio of 2:1,

ammonia solution as a precipitation agent and

oleic acid as a surfactant. In the process of

making salvigenin nanoparticles, the organic

phase was prepared by dissolving mPEG-b-

PLGA and Salvigenin in acetone. Fe3O4

solution was dissolved in different acetone.

Then the two solutions are mixed. After that

it was added to 20 mL of distilled water drop

by drop. After the addition of the complete

mixture, the acetone was removed under

vacuum with a rotary evaporator [23].

The characterization results showed that

Salvigenin loaded with mPEG-b-PLGA

coated with iron oxide nanoparticles had a

particle size of 57 ± 2 nm, a polydispersity

index of 0.168 ± 0.03, Zeta Potential 33 ± 1.2

mV and an encapsulation efficiency of 82 ±

1.6%. Nanoparticles are said to be stable and

positively charged based on the

characterization results [23].

3.19 Silibinin

Silibinin nanoparticles are prepared using the

nanoprecipitation method and utilizing

Eudragit and PVA. Eudragit was used as a

polymer and PVA was used as a stabilizer.

The reason for using Eudragit is because

Eudragit is a positive copolymer which has a

base site for ionized tertiary amine groups in

gastric juice which can make nanoparticles

easily soluble in the stomach [46].

Silibinin nanoparticles have the composition

Silibinin:Eudragit:PVA (1:10:10;w/w/w).

The organic phase was prepared by

dissolving Silibinin into ethanol in a closed

glass bottle. The aqueous phase contains

PVA. The characterization results showed

that Silibinin nanoparticles had a particle size

of 120 nm, Zeta Potential of 4.6 mV and

encapsulation efficiency of 79.0 ± 2.4%.

Small particle size < 200 nm so that drug

accumulation in tumor cells increases. The

positive charge is suitable for drugs that are

absorbed by charged cellular membranes

[46].

3.20 Silymarin

1. Silymarin-loaded lipid polymer

hybrid nanoparticles containing

chitosan

Silymarin was prepared into lipid-polymer

hybrid nanoparticles. This type of

nanoparticle utilizes liposomes and polymer

nanoparticles. These nanoparticles are

shaped like a shell core consisting of a

polymer core and a phospholipid wall. The

polymer used is chitosan. This is because

Chitosan has an amino group that can change

the charge to be positive when in a weak acid

state. The positive charge allows the

compound to adsorb mucin on the intestinal

R F. Pratama et al / Indo J Pharm 4 (2023) 307-332

328

mucosa through electrostatic interactions

[47].

The lipid-polymer hybrid of Silymarin

nanoparticles was prepared using a two-step

nanoprecipitation method. In this method,

lipid-polymer hybrid nanoparticles were

obtained by incubating the nanoparticle

dispersion with chitosan solution after

evaporation of the organic solvent. The

aqueous phase was prepared by dissolving

soybean lecithin and DSPE-PEG 2000 in

ethanol. The Organic Phase was prepared by

dissolving PLGA and silymarin in a mixture

of acetonitrile-methanol. Chitosan solution

was prepared by dissolving chitosan in 0.5%

acetic acid and then filtering through a 0.8 m

membrane [47].

After being characterized, the lipid-polymer

hybrid nanoparticles Silymarin with Chitosan

had a particle size of 286.5 ± 23.8 nm, a

polydispersity index of 0.226 ± 0.008, a Zeta

Potential of 45.3 ± 8.9 mV and an

encapsulation efficiency of 97.05 ± 0.01%.

The addition of chitosan increases the particle

size and changes the zeta potential charge

from negative to positive. In addition, the oral

bioavailability of silymarin in this form was

14.38 times higher than that of nanoparticles

without chitosan [47].

2. Silymarin-Loaded Eudragit

Silymarin is manufactured in the form of

Silymarin Loaded Eudragit Nanoparticles.

This is because Eudragit Polymer has the

ability to form positively charged

nanodispersions, moderate bio-adhesive

strength, and has no irritating effect on the

mucosal surface. Silymarin Loaded Eudragit

Nanoparticles were prepared by the

nanoprecipitation method. First, an organic

phase was made with a polymer composition

of silymarin and Eudragit with a constant

ratio of Eudragit RS 100 & Eudragit LS 100

(1:1 w/w) dissolved in acetone. The aqueous

phase having the composition of deionized

water containing PVA. PVA is a synthetic

polymer that is soluble in water and functions

as a stabilizer. Eudragit RS100 has very low

water permeability, while Eudragit RL100

has high water permeability. To improve the

quality of the drug release rate, these multiple

polymers were used concurrently [14].

In this study, nine formulations were made

with three different concentrations of PVA

(1, 2, 3% w/v) and three different

organic/water phase ratios (1:6, 1:10, 1:20

v/v). The optimal formulation chosen was a

drug/polymer ratio of 1:1 and stirring for 5

minutes at 480 rpm on a magnetic stirrer

followed by homogenization for 30 minutes

at 23.5 krpm. The optimal formulation has

characterization results with a particle size of

84.70 nm, a polydispersity index of 0.38 ±

0.01, and an encapsulation efficiency of

84.35 %. In addition, the oral bioavailability

of silymarin in this form was 14.38 times

higher than that of nanoparticles without

chitosan. When the PVA concentration was

increased, the particle size and polydispersity

index increased at low organic phase ratios to

water (1:6) while decreasing at high organic

phase ratios to water (1:10 and 1:20).

Moreover, at higher organic to water phase

ratio and as PVA concentration increased,

particle size and entrapment efficiency

decreased [14].

4. Conclusion

Based on the literature review that has been

done, there are various nanoprecipitation

methods that have been applied to the

preparation of flavonoid nanoparticles from

20 prepared secondary metabolites of

flavonoids. These methods consist of

Traditional Nanoprecipitation, Heat induced

evaporative antisolvent nanoprecipitation,

and Sonication-assisted nanoprecipitation.

The most frequently used method is

Traditional Nanoprecipitation.

The types of flavonoid nanoparticles which

were produced are polymeric nanoparticles,

lipid-polymer hybrid nanoparticles, magnetic

R F. Pratama et al / Indo J Pharm 4 (2023) 307-332

329

nanoparticles, organic-inorganic hybrid

nanoparticles and starch nanoparticles.

Polymeric nanoparticles are a type of

nanoparticle that is often produced.

Nanoparticles with the smallest particle size

were obtained from research on production of

Quercetin nanoparticles using PLA as a

carrier and PVA as a stabilizer using the

Sonication-assisted nanoprecipitation

method. PLGA in the manufacture of

Apigenin nanoparticles using the Traditional

Nanoprecipitation method produced the

lowest polydispersity index value. The use of

PVP and PCL in research on the manufacture

of Artocarpin, Naringenin and Floretin

nanoparticles resulted in an encapsulation

efficiency of greater than 99%.

Acknowledgements

Author of this article is grateful to the lecturer

who has been supportive and helpful in

providing data and information for the

purpose of this study.

A. K. Umar et al / Indo J Pharm 3 (2021) 57-70

330

References

[1] Kumar S, Pandey A. Chemistry and

Biological Activities of Flavonoids: An

Overview. The Scientific World Journal.

2013;2013:1-16.

[2] Ullah A, Munir S, Badshah S, Khan

N, Ghani L, Poulson B et al. Important

Flavonoids and Their Role as a Therapeutic

Agent. Molecules. 2020;25(22):5243.

[3] Modasiya MK, Patel JN, Rathod

DM, Patel NA. Techniques to improve the

solubility of poorly soluble drugs.

International Journal of Pharmacy and Life

Sciences. 2012;3(2):1459-1469

[4] Savjani KT, Gajjar AK, Savjani JK.

Drug solubility: importance and

enhancement techniques. ISRN Pharm.

2012;2012:195727.

doi:10.5402/2012/195727

[5] Barreras-Urbina CG, Ramírez-

Wong B, López-Ahumada GA, et al. Nano-

and Micro-Particles by Nanoprecipitation:

Possible Application in the Food and

Agricultural Industries, International

Journal of Food Properties. 2016;19:1912-

1923.

[6] Dobrzynska M, Napierala M,

Florek E. Flavonoid Nanoparticles: A

Promising Approach for Cancer Therapy.

Biomolecules. 2020;10(9):1268.

[7] Ansari MJ. Factors Affecting

Preparation and Properties gf Nanoparticles

by Nanoprecipitation Method. IAJPS.

2018;4(12):4854-4858.

[8] Massella D, Celasco E, Salaün F,

Ferri A, Barresi AA. Overcoming the

Limits of Flash Nanoprecipitation:

Effective Loading of Hydrophilic Drug into

Polymeric Nanoparticles with Controlled

Structure. Polymers (Basel).

2018;10(10):1092.

[9] Wang Y, Li P, Truong-Dinh Tran T,

Zhang J, Kong L. Manufacturing

Techniques and Surface Engineering of

Polymer Based Nanoparticles for Targeted

Drug Delivery to Cancer. Nanomaterials

(Basel). 2016;6(2):26.

[10] Awouafack MD, Tane P, Morita H.

Isolation and Structure Characterization of

Flavonoids. In: Justino, G. C. , editor.

Flavonoids - From Biosynthesis to Human

Health [Internet]. London: IntechOpen;

2017 [cited 2022 Jul 19]. Available from:

https://www.intechopen.com/chapters/545

24 doi: 10.5772/67881

[11] Kumar SR, Priyatharshni S, Babu

VN, et al. Quercetin conjugated

superparamagnetic magnetite nanoparticles

for in-vitro analysis of breast cancer cell

lines for chemotherapy applications. J

Colloid Interface Sci. 2014;436:234-242.

[12] Sechi M, Syed DN, Pala N, et al.

Nanoencapsulation of dietary flavonoid

fisetin: Formulation and in vitro antioxidant

and α-glucosidase inhibition activities.

Mater Sci Eng C Mater Biol Appl.

2016;68:594-602

[13] Zhao J, Yang J, Xie Y.

Improvement strategies for the oral

bioavailability of poorly water-soluble

flavonoids: An overview. Int J Pharm.

2019;570:118642.

doi:10.1016/j.ijpharm.2019.118642

[14] El-Nahas AE, Allam AN,

Abdelmonsif DA, El-Kamel AH.

Silymarin-Loaded Eudragit Nanoparticles:

Formulation, Characterization, and

Hepatoprotective and Toxicity Evaluation.

AAPS PharmSciTech. 2017;18(8):3076-

3086.

[15] Gaur PK. Nanosuspension of

flavonoid-rich fraction from Psidium

guajava Linn for improved type 2-diabetes

potential. Journal of Drug Delivery Science

and Technology. 2021;62:102358.

[16] Sharma G, Park J, Sharma AR, et al.

Methoxy poly(ethylene glycol)-

poly(lactide) nanoparticles encapsulating

quercetin act as an effective anticancer

agent by inducing apoptosis in breast

cancer. Pharm Res. 2015;32(2):723-735.

[17] Wathoni N, Nguyen AN, Rusdin A,

et al. Enteric-Coated Strategies in

Colorectal Cancer Nanoparticle Drug

Delivery System. Drug Des Devel Ther.

2020;14:4387-4405.

[18] Yao Y, Zhou Y, Liu L, et al.

Nanoparticle-Based Drug Delivery in

Cancer Therapy and Its Role in

R F. Pratama et al / Indo J Pharm 4 (2023) 307-332

331

Overcoming Drug Resistance. Frontiers in

Molecular Biosciences. 2020;7.

[19] Biriukov D, Fibich P, Předota M.

Zeta Potential Determination from

Molecular Simulations. The Journal of

Physical Chemistry C. 2020;124(5):3159-

3170

[20] Clogston JD, Patri AK. Zeta

potential measurement. Methods Mol Biol.

2011;697:63-70.

[21] Kharia A, Singhai A, Verma R.

Formulation and Evaluation of Polymeric

Nanoparticles of an Antiviral Drug for

Gastroretention. International Journal of

Pharmaceutical Sciences and

Nanotechnology. 2012;4:1557-1562.

[22] Lv Y, He H, Qi J, et al. Visual

validation of the measurement of

entrapment efficiency of drug nanocarriers.

Int J Pharm. 2018;547(1-2):395-403.

[23] Mansourizadeh F, Sepehri H,

Khoee S, Farimani M, Delphi L, Tousi M.

Designing Salvigenin –loaded mPEG-b-

PLGA @Fe3O4 nanoparticles system for

improvement of Salvigenin anti-cancer

effects on the breast cancer cells, an in vitro

study. Journal of Drug Delivery Science

and Technology. 2020;57:101619.

[24] Farrag Y, Ide W, Montero B, et al.

Preparation of starch nanoparticles loaded

with quercetin using nanoprecipitation

technique. Int J Biol Macromol.

2018;114:426-433.

[25] Krishnakumar N, Sulfikkaralia N,

RajendraPrasad N, Karthikeyan S.

Enhanced anticancer activity of naringenin-

loaded nanoparticles in human cervical

(HeLa) cancer cells. Biomedicine and

perventive nutrition. 2011.

[26] Wu B, Liang Y, Tan Y, et al.

Genistein-loaded nanoparticles of star-

shaped diblock copolymer mannitol-core

PLGA-TPGS for the treatment of liver

cancer. Mater Sci Eng C Mater Biol Appl.

2016;59:792-800.

[27] Ganguly S, Dewanjee S, Sen R, et

al. Apigenin-loaded galactose tailored

PLGA nanoparticles: A possible strategy

for liver targeting to treat hepatocellular

carcinoma. Colloids Surf B Biointerfaces.

2021;204:111778.

[28] Tzeng CW, Tzeng WS, Lin LT, Lee

CW, Yen FL, Lin CC. Enhanced

autophagic activity of artocarpin in human

hepatocellular carcinoma cells through

improving its solubility by a nanoparticle

system. Phytomedicine. 2016;23(5):528-

540.

[29] El-Hussien D, El-Zaafarany GM,

Nasr M, Sammour O. Chrysin

nanocapsules with dual anti-glycemic and

anti-hyperlipidemic effects: Chemometric

optimization, physicochemical

characterization and pharmacodynamic

assessment. Int J Pharm. 2021;592:120044.

[30] Al-Shalabi E, Alkhaldi M, Sunoqrot

S. Development and evaluation of

polymeric nanocapsules for cirsiliol

isolated from Jordanian Teucrium polium

L. as a potential anticancer nanomedicine.

Journal of Drug Delivery Science and

Technology. 2020;56.

[31] Dhas N, Mehta, TA. Intranasal

delivery of Chitosan decorated PLGA core

/shell nanoparticles containing flavonoid to

reduce oxidative stress in the treatment of

Alzheimer’s disease. Journal of Drug

Delivery Science and Technology.

2020;61.

[32] Meena R, Kumar S, Kumar R,

Gaharwar US, Rajamani P. PLGA-CTAB

curcumin nanoparticles: Fabrication,

characterization and molecular basis of

anticancer activity in triple negative breast

cancer cell lines (MDA-MB-231 cells).

Biomed Pharmacother. 2017;94:944-954.

[33] Dalcin A, Roggia I, Felin S,

Vizzotto B, Mitjans M, Vinardell M, et al.

UVB photoprotective capacity of hydrogels

containing dihydromyricetin nanocapsules

to UV-induced DNA damage. Colloids and

Surfaces B: Biointerfaces.

2021;197:11431.

[34] Freag M, Elnaggar Y, Abdallah O.

Development of novel polymer-stabilized

diosmin nanosuspensions: In vitro appraisal

and ex vivo permeation. International

journal of pharmaceutics. 2013; 454.

R F. Pratama et al / Indo J Pharm 4 (2023) 307-332

332

[35] Zhang H, Chen MK, Li K, Hu C, Lu

MH, Situ J. Eupafolin nanoparticle

improves acute renal injury induced by LPS

through inhibiting ROS and inflammation.

Biomed Pharmacother. 2017;85:704-711.

[36] Elmowafy M, Alhakimy NA,

Shalaby K, et al. Hybrid polylactic

acid/Eudragit L100 nanoparticles: A

promising system for enhancement of

bioavailability and pharmacodynamic

efficacy of luteolin. 2021;65.

[37] Kumar RP, Abraham A. PVP-

coated naringenin nanoparticles for

biomedical applications - In vivo

toxicological evaluations. Chem Biol

Interact. 2016;257:110-118.

[38] Casarini TPA, Frank LA, Benin T,

Onzi G, Pohlmann AR, Guterres SS.

Innovative hydrogel containing polymeric

nanocapsules loaded with phloretin:

Enhanced skin penetration and adhesion.

Mater Sci Eng C Mater Biol Appl.

2021;120:111681.

[39] Yi T, Huang J, Chen X, Xiong H,

Kang Y, Wu J. Synthesis, characterization,

and formulation of poly-puerarin as a

biodegradable and biosafe drug delivery

platform for anti-cancer therapy.

Biomaterials Science. 2019;7.

[40] Fawzy AS, Priyadarshini BM,

Selvan ST, Lu TB, Neo J.

Proanthocyanidins-Loaded Nanoparticles

Enhance Dentin Degradation Resistance. J

Dent Res. 2017;96(7):780-789.

[41] Pandey SK, Patel DK, Thakur R,

Mishra DP, Maiti P, Haldar C. Anti-cancer

evaluation of quercetin embedded PLA

nanoparticles synthesized by emulsified

nanoprecipitation. Int J Biol Macromol.

2015;75:521-529.

[42] Thakur NS, Mandal N, Patel G, et

al. Co-administration of zinc

phthalocyanine and quercetin via hybrid

nanoparticles for augmented photodynamic

therapy. Nanomedicine. 2021;33:102368.

[43] Sunoqrot S, Abujamous L. pH-

sensitive polymeric nanoparticles of

quercetin as a potential colon cancer-

targeted nanomedicine. Journal of Drug

Delivery Science and Technology.

2019;52:670-676.

[44] Kumar V. Verma P, Singh S.

Development and evaluation of

biodegradable polymeric nanoparticles for

the effective delivery of quercetin using a

quality by design approach. LWT - Food

Science and Technology. 2015;61.

[45] Asfour MH, Mohsen AM.

Formulation and evaluation of pH-sensitive

rutin nanospheres against colon carcinoma

using HCT-116 cell line. J Adv Res.

2017;9:17-26.

[46] Gohulkumar M, Gurushankar K,

Rajendra Prasad N, Krishnakumar N.

Enhanced cytotoxicity and apoptosis-

induced anticancer effect of silibinin-

loaded nanoparticles in oral carcinoma

(KB) cells. Mater Sci Eng C Mater Biol

Appl. 2014;41:274-282.

[47] Liang J, Liu Y, Liu J, et al.

Chitosan-functionalized lipid-polymer

hybrid nanoparticles for oral delivery of

silymarin and enhanced lipid-lowering

effect in NAFLD. Journal of

Nanobiotechnology. 2018;16.

[48] Liu Y, et al. Formulation of

Nanoparticles Using Mixing-Induced

Nanoprecipitation for Drug Delivery. Ind.

Eng. Chem. Res. 2020;59(9): 4134–4149.

[49] Bosselmann S, Nagao M, Chow K

T, et al. Influence of formulation and

processing variables on properties of

itraconazole nanoparticles made by

advanced evaporative precipitation into

aqueous solution. AAPS PharmSciTech.

2012;13(3):949–960.

[50] Mugheirbi N, Paluch K, Tajber L.

Heat induced evaporative antisolvent

nanoprecipitation (HIEAN) of

itraconazole. International journal of

pharmaceutics. 2014.

[51] Baldassarre F, et al. Sonication-

Assisted Production of Fosetyl-Al

Nanocrystals: Investigation of Human

Toxicity and In Vitro Antibacterial

Efficacy against Xylella Fastidiosa.

Nanomaterials. 2020;10(6):1174.

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