A REVIEW ON RECENT ADVANCEMENT IN THE METHOD OF ...

www.wjpps.com Vol 5, Issue 7, 2016.

351

Singh et al. World Journal of Pharmacy and Pharmaceutical Sciences

A REVIEW ON RECENT ADVANCEMENT IN THE METHOD OF

PREPARATION OF NENOPARTICLES FOR THE ANTIMICROBIAL

BROAD SPECTRUM

Pawan Singh*1 and Prevesh Kumar

2

Assistant Professor, Department of Pharmacy Academy, IFTM University, Moradabad.

ABSTRACT

In the recent advancing trend in drug delivery system by nanoparticles.

In this trend the polymer and antibiotics drug delivery. The drug

stability and drug interaction is more important factor for a control and

perfect dug delivery. The achieving higher dose drug delivery in

systemic circulation and observed the higher systemic effect with the

lesser amount producing less side effect as compare other drug

delivery system. While large numbers of preclinical studies have been

published, the emphasis here is placed on preclinical and clinical

studies that are likely to affect clinical inquiries and their implications

for proceeding the treatment of patients with cancer and microbial

infection.

KEYWORDS: Nanoparticle, method of preparation, antimicrobial spectrum.

INTRODUCTION

Drug delivery System: Drug delivery is the method for administering a pharmaceutical

compound to achieve a therapeutic effect in humans or animals. Drug delivery system can

have very important role in efficacy of drugs.[1]

some drugs have an optimum concentration

of range within which maximum effect is derived. But there is very slow progress in efficacy

of the action of severe syndrome, has suggest a developing need of drug delivery system.[2]

Drug delivery system is multi-disciplinary approach to delivery of therapeutics to the target

tissue which gives new ideas on controlling the pharmacokinetics, pharmacodynamics,

immunogenicity, bio recognition, nonspecific toxicity and efficacy of the drug.[3]

Drug

delivery system (DDS) are based on interdisciplinary methodology that combine polymer

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.041

Volume 5, Issue 7, 351-374. Review Article ISSN 2278 – 4357

*Corresponding Author

Pawan Singh

Assistant Professor,

Department of Pharmacy

Academy, IFTM

University, Moradabad.

Article Received on

08 May 2016,

Revised on 29 May 2016,

Accepted on 18 June 2016,

DOI: 10.20959/wjpps20167-6891

http://www.wjpps.com/


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science, pharmaceutics, molecular biology and bio conjugate chemistry. The main goal of

every drug delivery is to deliver the precise amount of drug on aim area. An essential guide

for biomedical engineers and pharmaceutical designers, this resource combines

physiochemical principles with physiological process to facilitate the design of the system

that will deliver drug at target area with exact time.[4]

The main approach of drug delivery

system is to promoting the exposure of drug on targeted area rather than non-target area to

avoid unnecessary side effects.

Novel drug delivery system is based on two mechanisms:

1. Physical mechanism

2. Biochemical mechanism

In physical mechanism include osmosis, diffusion, and erosion.

And in biochemical mechanism include monoclonal antibiotics, gene therapy, and vector

system. Drug delivery system (DDS) such as biodegradable polymer based nanoparticles can

be designed to improve drug bioavailability orally. There are many antibiotics, antifungal,

anticancer drugs which are recover by different drug delivery systems. DDS are designed to

alter the pharmacokinetics and bio circulation of the drug.[5]

The oral route remains the preferred to administrate drugs, but due to their physicochemical

and enzymatic barriers, they still have to be administered parentally. To overcome this, the

parental drugs release has been studied since few decades, which lead to the improvement of

numerous system, allowing an efficient system of compound to control release.[6]

Many types of drug delivery systems are in various stages of examination. These particles

have been architectures in such a way to ultimately lead to control and efficient release. Most

of the current research is mainly focusing on using nanoparticles as drug delivery carriers for

challenging to treat infectious and life binding syndrome.[7]

Using nanoparticles to carry

drug at target site lead to effective and efficient result.

Polymeric Nanoparticles and its classification

Conservative preparations like solution, suspension or emulsion suffer from certain limitations

like high dose and low availability, first pass effect, intolerance, instability, and they exhibit

fluctuations in plasma drug levels and do not provide sustained effect,[8]

therefore there is a



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need for some novel carriers which could meet ideal requirement of drug delivery system.

Recently nanoparticles delivery system has been proposed as colloidal drug carriers.[9]

Polymeric Nanoparticles (PNP) are defined as particulate dispersion or solid particles with a

size range of 10 to 1000 nm in diameter.[10]

The term PNP is a collective term given for any

type of polymer nanoparticle, but specifically for nanospheres and nanocapsules.

Nano spheres are matrix particles, i.e., particles whose entire mass is solid and molecules may

be adsorbed at the sphere surface or encapsulated within the particle. In general, they are

spherical, but ―nanospheres‖ with a nonspherical shape are also described in the literature.[11]

Nanocapsules are vesicular systems, acting as a kind of reservoir, in which the entrapped

substances are confined to a cavity consisting of a liquid core (either oil or water)

surrounded by a solid material shell.[12]

Nanoparticles may or may not exhibit size-related

properties that differ significantly from those observed in fine particles or bulk materials.

The major goal in designing of polymeric nanoparticles as a delivery system is,

1. To control particles size

2. Surface property

3. Release of pharmaceutical active agent in order to achieve in site design of drug at

therapeutically optical range and dose regimen

Drug release from nanoparticles

The nanoparticle is coated by polymer, which releases the drug by controlled diffusion or

erosion from the core across the polymeric membrane or matrix.[13]

The membrane coating

acts as a barrier to release, therefore, the solubility and diffusivity of drug in polymer

membrane becomes the determining factor in drug release.[14]

Furthermore release rate can

also be affected by ionic interaction between the drug and addition of auxiliary ingredients.

When the drug is involved in interaction with auxiliary ingredients to form a less water

soluble complex, then the drug release can be very slow with almost no burst release

effect.[15]

To develop a successful Nano particulate system, both drug release and polymer

biodegradation are important consideration factors.

In general, drug release rate depends on

(1) Solubility of drug, (2) Desorption of the surface bound/ adsorbed drug, (3) Drug diffusion

through the nanoparticle matrix, (4) Nanoparticle matrix erosion/degradation and (5)



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Combination of erosion/diffusion. Thus solubility, diffusion and biodegradation of the matrix

materials govern the release process

Preparation of biodegradable polymeric nanoparticles

The selection of appropriate method for the preparation of nanoparticles depends on the

physicochemical properties of the polymer and the drug to be loaded. The primary

manufacturing methods of nanoparticles from preformed polymer includes:

1. Emulsion solvent evaporation method.

2. Double Emulsion and Evaporation Method.

3. Salting out method.

4. Emulsion diffusion method.

5. Solvent displacement/precipitation method.

6. Ionic gelation method.

There are many other modified techniques used for preparation of biodegradable nanoparticles.

The key advantages of nanoparticles over other Nano carriers

1. Improved bioavailability by enhancing aqueous solubility,

2. Increasing resistance time in the body (increasing half-life for clearance/increasing

specificity for its cognate receptors.

3. Targeting drug to specific location in the body (its site of action).[16]

This results in

concomitant reduction in quantity of the drug required and dosage toxicity, enabling the

safe delivery of toxic therapeutic drugs and protection of non-target tissues and cells

from severe side effects. It is increasingly used in different submissions, including drug

carrier systems and to pass organ barriers such as the blood-brain barrier, cell membrane

etc. They are based on biocompatible lipid and provide sustained effect by either diffusion

or dissolution.[17]

4. When compared to single unit operation, multi-particulate systems such as nanoparticles

distributes more uniformly in GIT. Resulting in more uniform absorption and a bridged

danger of local irritation.

5. Nanoparticles shown to be more stable than liposomes in biological fluids and if

endocytosis of intact liposomes by the intestinal cells occurs at all, it remains a rare

event, thus limiting the potential applications of these carriers.[18]

The aim of drug



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targeting is to deliver drugs to the right place, at the high concentration, for the right

period of time.

Bacterial Infections

Bacteria are microscopic, single-celled organisms. There are thousands of different kinds, and

they live in every conceivable environment all over the world.[19]

They aware in soil,

seawater, and deep within the earth’s crust. Particular bacteria have been informed even to

live in radioactive waste. Many bacteria live in the bodies of people and animals—on the

skin and in the airways, mouth, and digestive, reproductive, and urinary tracts—without

affecting slightly detriment. Such bacteria are called occupier flora or the micro-biome.[20]

Many resident flora are actually helpful to people—for example by helping them digest food

or by preventing the growth of other, more hazardous bacteria.

Only a few kinds of bacteria cause disease. They are called pathogens. Sometimes bacteria

that normally reside inoffensively in the body cause disease. Bacteria can cause syndrome by

producing harmful substances (toxins), invading tissues, or doing both.[21]

Classification

The classification of bacteria serves a variety of different functions. Because of this variety,

bacteria may be grouped using many different typing schemes. The critical feature for all

these classification systems is an organism identified by one individual (scientist, clinician,

epidemiologist), is recognized as the same organism by another individual. At present the

typing schemes used by clinicians and clinical microbiologists rely on phenotypic typing

schemes. These schemes utilize the bacterial morphology and staining properties of the

organism, as well as O2 growth requirements of the species combined with a variety of

biochemical tests. For clinicians, the environmental reservoir of the organism, the vectors and

means of transmission of the pathogen are also of great importance. The classification

schemes most commonly used by clinicians and clinical microbiologists are discussed below.

Scientists interested in the evolution of microorganisms are more interested in taxonomic

techniques that allow for the comparison of highly conserved genes among different species.

A relatively new application of this technology has been the recognition and characterization

of non-cultivatable pathogens and the diseases that they cause.



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Phenotypic classification systems

Gram stain and bacterial morphology: Of all the different classification systems, the Gram

stain has withstood the test of time. Discovered by H.C. Gram in 1884 it remains an

important and useful technique to this day.[22]

It allows a large proportion of clinically

important bacteria to be classified as either Gram positive or negative based on their

morphology and differential staining properties. Slides are sequentially stained with crystal

violet, iodine, then destained with alcohol and counter-stained with safranin.[23]

Gram positive

bacteria stain blue-purple and Gram negative bacteria stain red. The difference between the

two groups is believed to be due to a much larger peptidoglycan (cell wall) in Gram positives.

As a result the iodine and crystal violet precipitate in the MID 1 thickened cell wall and are

not eluted by alcohol in difference with the Gram negatives where the crystal violet is readily

eluted from the bacteria. As a result bacteria can be famous based on their morphology and

staining properties.[24]

Some bacteria such as mycobacteria (the cause of tuberculosis) are not reliably stained due to

the large lipid content of the peptidoglycan. Alternative staining techniques (Kinyoun or acid

fast stain) are therefore used that take advantage of the resistance to de-staining after

lengthier initial staining. Bacteria can be classified in several ways:

Scientific names

Bacteria, like other living things, are classified by genus (based on having one or several

similar characteristics) and, within the genus, by species. Their scientific name is genus

followed by species (for example, Clostridium botulinum). Within a species, there may be

different types, called strains. Strains differ in genetic makeup and chemical components.

Sometimes certain drugs and vaccines are effective only against certain strains.

Staining

Bacteria may be classified by the color they turn after certain chemicals (stains) are applied

to them. A commonly used stain is the Gram stain. Some bacteria stain blue. They are called

gram-positive. Others stain red. They are called gram-negative. Gram-positive and gram-

negative bacteria stain inversely because their cell walls are different. They also cause

different types of infections, and different types of antibiotics are effective against them.



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Shapes

All bacteria may be classified as one of three basic shapes: spheres (cocci), rods (bacilli), and

spirals or helixes (spirochetes).

Fig: 1 shape of bacteria’s

Need for oxygen

Bacteria are also classified by whether they need oxygen to live and grow. Those that need

oxygen are called aerobes. Those that have trouble living or growing when oxygen is present

are called anaerobes. Some bacteria, called facultative bacteria, can live and grow with or

without oxygen.

Distinguishing Features between Gram Positive and Negative Bacteria

Gram positive bacteria have a large peptidoglycan structure. As noted above, this accounts for

the differential staining with Gram stain. Some Gram positive bacteria are also capable of

forming spores under stressful environmental conditions such as when there is limited

availability of carbon and nitrogen.[25]

Spores therefore allow bacteria to survive exposure to

extreme conditions and can lead to re-infection (e.g., pseudomembranous colitis from

Clostridium difficle).

Gram negative bacteria have a small peptidoglycan layer but have an additional membrane,

the outer cytoplasmic membrane. This creates an additional permeability barrier and results

in the need for transport mechanisms across this membrane.[26]

A major component of the cytoplasmic membrane that is unique to Gram negatives is

endotoxin. This component is essential for bacterial survival. Endotoxin has three



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components: the lipid A moiety, the highly conserved core polysaccharide, and the

species specific O antigen (also polysaccharide). In contrast with the secreted exotoxins,

endotoxin is cell-associated but can be released during cell division or cell death.[27]

The

Lipid A moiety of endotoxin is responsible for sepsis which may be fatal. Sepsis is

characterized clinically by confusion, fever, drop in blood pressure and ultimately multi-

organ failure.

Gram Positive Bacteria

Name Morphology O2

Requirements Commensal

Reservoirs/ Sites of

colonization,

Transmission

Types of

Infections

Staphylococci Cocci in grape

like clusters

facultative

anaerobe Yes

Skin, nares/endogenous,

direct contact, aerosol

Soft tissue, bone,

joint, endocarditis,

food poisoning

Streptococci

Cocci in pairs, chains

Facultative

anaerobe

Some

species

Oropharynx, skin/

endogenous, direct

contact, aerosol

Skin, pharyngitis,

endocarditis, toxic

shock

Pneumococci Diplococci,

lancet shaped

Facultative

anaerobe ±

Oropharynx, sinus /

aerosol

Pneumonia, otitis,

sinusitis,

meningitis

Enterococci Cocci in

pairs, chains

Facultative

anaerobe Yes

GI tract/ endogenous,

direct contact

UTI, GI,

catheterrelated

infections

Bacilli Rods,

sporeforming aerobic ±

Soil,air,water, animals

/aerosol, contact

Anthrax, food

poisoning,

catheterrelated

infections

Clostridia Rods, spore

formers anaerobic

Some

species

GI tract, soil/Breach

of

Tetanus, diarrhea,

gas

skin,endogenous,

ingestion

gangrene,

botulism

Corynebacterium Rods, nonspore

forming

Facultative

anaerobe

Some

species

Skin Catheter- related

infections, diphtheria

Listeria

Meningitis

Rods,

nonspore formers

facultative

anaerobe No

Animals, food products

/ Meningitis

Actinomyces

Irregular,

filamentous, form

sulfur granules

anaerobic Yes GI tract/ endogenous Skin, soft tissue

Gram Negative Bacteria

Name Morphology O2


Reservoirs /Sites of

colonization, Tr

ansmission

Types of

Infections

Enterobacteria

ceae (E. coli, Rods

facultative

anaerobe

Some

species

GI tract, animals/

Endogenous, fecaloral

Diarrhea, urinary

tract, food



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klebsiella,

salmonella,

shigella)

poisoning, sepsis

Bacteroides Rods anaerobic Yes GI tract/ Endogenous Abscesses,

intraabdomin al

infections

Pseudomonas Rods aerobic No

Water, soil/

Endogenous, breach of

skin barrier

Infections in

immunocomp ro

mised hosts,

Cystic Fibrosis

Vibrio (cholera) Rods, curved

shape

microaerophil

ic No

Water / Contaminated

food, water Diarrhea

Campylobacter Rods, curved

shape

microaerophil

ic No

Food / Ingestion

of contaminated food

Diarrhea,

Bacteremia

Legionella Rods, poorly

stained

microaerophil

ic No

Water/Inhalation of

aerosol

Pneumonia,

febrile illness

Neisseria Cocci, kidneybean

shaped Microaerophilic

No (N.

meningitidis

sometimes)

Humans/ Sexual,

aerosol

Meningitis,

pelvic

inflammatory

disease

Hemophilus Coccobacillary

- pleomorphic

Facultative

anaerobe

Some

species

Respiratory

tract/Endogenous,

aerosol

Respiratory,

sinusitis, otitis

meningitis

Bartonella Small,

pleomorphic rods

aerobic/

microaerophilic No

Cats, fleas, lice / cat

bites, lice or fleas?

Cat scratch

disease,

endocarditis,

bacillary

angiomatosis

Miscellaneous Bacteria

Name Morphology O2


Reservoirs/Sites of

colonization,

Transmission

Types of

Infections

Helicobacter

GN, but not

visible on Gram

stain - helical

(corkscrew)

shaped

microaerophilic Yes Stomach/ Endogenous,

Fecal-oral

peptic ulcer

disease, gastric

ulcer

Mycobacteria

Rods, Weakly

Gram positive,

Acid fast stain

positive

aerobic No Lungs/Fomites Tuberculosis

Treponemes

Not visible on

Gram stain, spiral

shaped on dark

field exam

nonculturable

on

routine media

No Humans/ Sexual

transmission Syphilis



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Borrelia

Not visible on

Gram stain, spiral

shaped on dark

field exam

nonculturable

on routine

media

No Rodents, Ticks/ Tick

bites

Lyme, Relapsing

fever

Mycoplasma

Not visible on

Gram stain, no

cell wall,

pleomorphic

Non-culturable

on routine

media

Some

species Humans / aerosol

Respiratory tract

infections

Rickettsia/

Ehrlichia

Obligate

Intracellular

(Gram negative

but not visible on

Gram Stain)

Non-culturable

on routine

media

No

Ticks, Mites/transmitted

from the feces of

infected lice, fleas, ticks

Cause a variety

of illnesses

including

systemic

vasculitis

(e.g. Rocky

Mountain

Spotted Fever),

rash, pneumonia

Bacterial Defenses

Bacteria have many ways of defending themselves.

Biofilm

Some bacteria secrete a substance that helps them attach to other bacteria, cells, or objects.

This substance combines with the bacteria to form a sticky layer called biofilm. For example,

certain bacteria form a biofilm on teeth (called dental plaque). The biofilm traps food

particles, which the bacteria process and use, and in this process, they probably cause tooth

decay. Biofilms also help protect bacteria from antibiotics.

Capsules

Some bacteria are enclosed in a protective capsule. This capsule helps prevent white blood

cells, which fight infection, from ingesting the bacteria. Such bacteria are described as

encapsulated.

Outer membrane

Under the capsule, gram-negative bacteria have an outer membrane that protects them against

certain antibiotics. When disrupted, this membrane releases toxic substances called

endotoxins. Endotoxins contribute to the severity of symptoms during infections with gram-

negative bacteria.



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Spores

Some bacteria produce spores, which are an inactive (dormant) form. Spores can enable

bacteria to survive when environmental conditions are difficult. When conditions are

favorable, each spore germinates into an active bacterium.

Flagella

Flagella are long, thin filaments that protrude from the cell surface and enable bacteria to

move. Bacteria without flagella cannot move on their own.

Antibiotic resistance

Some bacteria are naturally resistant to certain antibiotics.

Other bacteria develop resistance to drugs because they acquire genes from other bacteria that

have become resistant or because their genes mutate.[28]

For example, soon after the drug

penicillin was introduced in the mid-1940s, a few individual Staphylococcus aureus bacteria

acquired genes that made penicillin ineffective against them.[29]

The strains that possessed

these special genes had a survival advantage when penicillin was commonly used to treat

infections. Strains of Staphylococcus aureus that lacked these new genes were killed by

penicillin, allowing the remaining penicillin-resistant bacteria to reproduce and over time

become more common.[30]

Chemists then altered the penicillin molecule, making a different

but similar drug, methicillin, which could kill the penicillin-resistant bacteria. Soon after

methicillin was introduced, strains of Staphylococcus aureus developed genes that made them

resistant to methicillin and related drugs. These strains are called methicillin-resistant

Staphylococcus aureus (MRSA).

The genes that encode for drug resistance can be passed to following generations of bacteria

or sometimes even to other species of bacteria.

The more often antibiotics are used, the more likely resistant bacteria are to develop.

Therefore, doctors try to use antibiotics only when they are necessary. Giving antibiotics to

people who probably do not have a bacterial infection,[31]

such as those who have cough and

cold symptoms, does not make people better but does help create resistant bacteria. Because

antibiotics have been so widely used (and misused), many bacteria are resistant to certain

antibiotics.



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Resistant bacteria can spread from person to person. Because international travel is so

common, resistant bacteria can spread to many parts of the world in a short time. Spread of

these bacteria in hospitals is a particular concern.[32]

Resistant bacteria are common in

hospitals because antibiotics are so often necessary and hospital personnel and visitors may

spread the bacteria if they do not strictly follow appropriate sanitary procedures. Also, many

hospitalized patients have a weakened immune system, making them more susceptible to

infection.[33]

Resistant bacteria can also spread to people from animals. Resistant bacteria are common

among farm animals because antibiotics are often routinely given to healthy animals to

prevent infections that can impair growth or cause illness.[34]

METHODS OF PREPARATION NANOPARTICLES FOR ANTIMICROBIAL

ACTION

Ionotropic gelation method

Ionotropic gelation is based on the ability of polyelectrolytes to cross link in the presence of

counter ions to form hydrogel beads also called as gelispheres. Gelispheres are spherical

crosslinked hydrophilic polymeric entity capable of extensive gelation and swelling in

simulated biological fluids and the release of drug through it controlled by polymer

relaxation.[35]

The hydrogel beads are produced by dropping a drug-loaded polymeric

solution into the aqueous solution of polyvalent cations.[36]

The cations diffuses into the

drug-loaded polymeric drops, forming a three dimensional lattice of ionically crosslinked

moiety. Biomolecules can also be loaded into these gelispheres under mild conditions to retain

their three dimensional structure .[37]

Polyelectrolyte solution

[Sodium Alginate (-)/Gellan gum (-)/CMC (-)/Pectin (-)/ Chitosan (+) + Drug]

↓

Added drop wise under magnetic stirring by needle

↓

Counter ion solution

[Calcium chloride solution (+)/Sodium tripolyphosphate (-)]

↓

Gelispheres



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In Ionotropic gelation technique, there has been a growing interest in the use of natural

polymers as drug carriers due to their biocompatibility and biodegradability.[38]

The natural

or semisynthetic polymers i.e. Alginates, Gellan gum, Chitosan, Pectin and Carboxymethyl

cellulose are widely use for the encapsulation of drug by this technique.[39]

These natural

polyelectrolytes contain certain anions/cations on their chemical structure, these

anions/cations forms meshwork structure by combining with the counter ions and induce

gelation by cross linking. In spite of having a property of coating on the drug core these

natural polymers also acts as release rate retardant.[40]

Ionic gelation technique presents the following advantages over other methods:

The nanoparticles are obtained spontaneously under mild control conditions without

involving high temperatures, organic solvents, or sonication. and TPP is a multivalent

polyanion, with low toxicity and cost, unlike other cross-linkers, it presents no severe

constraints of handling and storage. After adding TPP solution, nanoparticles form

immediately through inter and intramolecular linkages created between TPP phosphates and

EXP amino groups.

Natural polymers used in ionotropic gelation method- Alginates

Alginate is a non-toxic, biodegradable, naturally occurring polysaccharide obtained from

marine brown algae, certain species of bacteria. (41)Sodium alginate is a sodium salt of

alginic acid a natural polysaccharide and a linear polymer composed of 1,4-linked β-D-

Mannuronic acid (M) and α-D-gluronic acid (G) residues in varying proportions and

arrangements.[42]

Sodium alginate is soluble in water and form a reticulated structure which

can be cross-linked with divalent or polyvalent cations to form insoluble meshwork. Calcium

and zinc cations have been reported for cross-linking of acid groups of alginate.[43]

Gellan gum

Gellan gum is a bacterial exopolysaccharide prepared commercially by aerobic submerged

fermentation of Sphingomones Eloda.[44]

A concentrated water solution of gellan gum is

made warm up preliminary to induce the gellan gelation. When the temperature is decreased,

the chains undergo a conformational transition from random coils to double helicles (coil-

helix transition).[45]

Then rearrangement of a double helicles occurs leading to the formation

of ordered junction zones (sol-gel transition), thus giving a thermo-reversible hydrogel.[44]



364


Chitosan

Chitosan is natural poly-(aminosaccharide), having structural characteristics similar to

glycosaminoglycans, is non-toxic and easily bioabsorbable.[46]

Chitosan due to its antacid

and antiulcer characteristics prevents or weakens drug irritation in the stomach.[47]

Chitosan

is a biopolymer which could be used for the preparation of various polyelectrolyte complex

products with natural polyanions such as xanthan, alginate, and carrangeenan.[48]

Among

these, complexes, chitosan-alginate complex may be the most important drug delivery

hydrogel system.

Carboxymethyl cellulose

The cellulose, a plant product on carboxymethylation process, can be modified as

carboxymethylcellulose (CMC).[49]

The interactions of the carboxylic groups of the CMC

with multivalent metal ions can be used to form so called ionotropic gels, which are

predominantly stabilized by the electrostatic interactions.[50]

In addition, interactions between

the –OH groups of the polymer and the metal ions contribute to the stability and the water

insolubility of these polymeric aggregates. The CMC can be cross-linked with

ferric/aluminum salt to get biodegradable hydrogel beads.[51]

Controlled release pattern can also be improved by coating these hydrogels with

chitosan/gelation and by cross-linking.

Pectin

Pectin is an inexpensive, non-toxic polysaccharide extracted from citrus peels or apple

pomaces, and has been used as a food additive,[52]

a thickening agent and a gelling agent.

Basically, it is a polymer of a-D-galacturonic acid with 1-4 linkages.

Factors affecting ionotropic gelation method[53]

1) Polymer and crosslinking electrolyte concentration

Polymer and electrolyte concentration have major effect on formulation of beads by

ionotropic gelation method. Concentration of both should in the ratio calculated from number

of crosslinking units. Percent entrapment efficiency varies from the type of electrolytes

and also the concentration of electrolytes.



365


2) Temperature

Temperature also plays imp role on size of beads formed by ionotropic gelation method and

also on the curing time i.e. time required for crosslinking.

3) pH of crosslinking solution

pH of crosslinking solution also considerable factor during the formulation as it shows effect

on reaction rate, shape and size of beads.

4) Drug concentration

Drug to be entrapped in the beads should be in the proper ratio with the polymer, as the drug

concentration greatly affects the entrapment efficiency, if drug: polymer ratio exceeds the

range then bursting effect may observe, density of gelispheres enhances and the size and

shape of gelispheres also increases.

5) Gas forming agent concentration

Gas forming agents such as calcium carbonate, sodium bicarbonate added in to the

formulation to develop porous gelispheres, which tremendously affect the gelispheres size

and shape. As gas forming agent forms porous gelispheres, breaks the lining of gelispheres

and results into the irregular surface.

Advances in Ionotropic gelation[54]

1) Polyelectrolyte complexation technique/ Ionotropic pre-gelation

The quality of hydrogel beads prepared by ionotropic gelation method can also be further

improved by polyelectrolyte complexation technique.[53]

The mechanical strength and

permeability barrier of hydrogels can be improved by the addition of oppositely charged

another polyelectrolyte to the ionotropically gelated gelispheres. For instance, addition of

polycations allows a membrane of polyelectrolyte complex to form on the surface of alginate

gelispheres. Authors Anil K. Anal, Willem F. Stevens reported a method for polyelectrolyte

beads of ampicillin prepared by ionotropic gelation method. Authors selected alginate and

chitosan for complexation and reported enhancement in encapsulation efficiency and

improved properties of controlled release of formed multilayer ampicillin.

2) Ionotropic gelation under a high voltage electrostatic field

Authors Lihua Ma and Changsheng Liu reported a modified ionotropic gelation method by

combining it with a high voltage electrostatic field to prepare protein-loaded chitosan



366


microspheres. This is new method for sustain delivery of Bovine Serum Albumin (BSA) by

encapsulating in chitosan microsphere also reported that the microspheres exhibited good

sphericity and dispersibility when the mixture of sodium tripolyphosphate (TPP) and ethanol

was applied as coagulation solution.[55]

The results from the literature survey suggest that

ionotropic gelation method combined with a high voltage electrostatic field is an effective

method for sustained delivery of protein by gelispheres.

3) Emulsion-internal ionotropic gelation

It is the advanced method in ionotropic gelation with the incorporation of oily phase and

emulsifier. As reported by Singla and colleagues the dispersed phase consisting of 40 mL of

2% v/v aqueous acetic acid containing 2.5% w/v chitosan was added to the continuous phase

consisting of hexane (250 mL) and Span 85 (0.5% w/v) to form a w/o emulsion. After 20

minutes of mechanical stirring, 15 mL of 1N sodium hydroxide solution was added at the rate

of 5mL per min at 15min intervals. Stirring speed of 2000 to 2200 rpm was continued for 2.5

hours. The microspheres were separated by filtration and subsequently washed with

petroleum ether, followed by distilled water and then air dried.[56]

Also Anita G. Sullad, Lata

S. Manjeshwar and Tejraj M. Aminabhav developed microspheres of Abacavir sulfate by w/o

emulsion method using Carboxy methyl guar gum, an anionic synthetic derivative.

Author Deepak singh and his colleagues developed Dry Powder Inhalation system of

Terbutaline sulfate for management of Asthma and the microspheres of Terbutaline sulfate

prepared by emulsification-ionotropic gelation and heat crosslinking agent. According to this

method aqueous solutions of chitosan and Terbutaline sulfate (in 0.5% acetic acid) were

emulsified in oil phase (100-200ml) consisting of dichloromethane and light liquid paraffin

(LLP) using homogenizer for 15 min. Span 80 was used as an emulsifier and lecithin as a co-

emulsifier and deaggregating agent. Cross-linking solution (citric acid, tripolyphosphate and

glucose 1%; 5-15 ml) was added to this emulsion and homogenization was continued for

another 30 min. This emulsion was then added slowly to light liquid paraffin (50 ml) which

was previously heated and maintained at 120° ± 10°C with continuous stirring for another one

hour. The hot oily dispersion of microspheres was then allowed to cool to room temperature

with continuous stirring at same speed, and finally centrifuged on a high-speed centrifuge at

10000 rpm for 10 min, in order to separate the microspheres. The sediment was dispersed in

diethyl ether to remove the oil, and this dispersion was again centrifuged for 3 min at the same

speed. Washing with diethyl ether was repeated three more times in a similar manner to



367


remove traces of oil. The sediment thus obtained was dried in oven at 50°-60°C, passed

through 100-mesh sieve and stored.

w/o/w emulsion solvent evaporation containing ionotropic gelation is the modified technique

involving multiphase. This method draws more attention nowadays, as the method is useful

for encapsulation of water-soluble drugs, proteins, DNA or antigens into microsphere or

nanosphere as effective delivery carriers.

4) Ionotropic gelation followed by coacervation

Jaejoon Han, Anne-sophie Guenier and colleagues successfully developed a new

encapsulation method involving two polymers (alginate and chitosan) and using methods of

functionalization (acylation) and ionotropic gelation followed coacervation to improve the

stability and physicochemical properties of beads. Beads were formed by ionotropic gelation

via calcium crosslinking and by alginate-chitosan complex coacervation. The main

difference between native and functionalized beads consisted in the presence of fatty acid

chains in the core (palmitoylated alginate) and external layer (palmitoylated chitosan) of

beads. Hence, alginate cross-links improved insolubility of beads by ionotropic gelation and

alginate-chitosan coacervation, which led to polyionic links between the core bead and the

external layer. Functionalization increases hydrophobic interactions into polymeric matrix

involving structural changes also improves the polymers barrier property by decreasing water

uptake and Water vapour pressure. Functionalized polymers did not improve their mechanical

properties and stability of micronutrients encapsulated in native and functionalized beads.

Authors also demonstrated that encapsulation had an excellent capacity to protect bioactive

molecules against temperature, humidity, and acidic conditions and allowed a controlled

release of these compounds during gastrointestinal transit.

C.L. Gerez, G. Font de Valdez and colleagues also developed novel microencapsulation of

Lactobacillus rhamnosus by ionotropic gelation using pectin (PE) and pectin-whey protein

(PE- WP). Both types of beads were covered with a layer of whey protein by complex

coacervation to improve the survival rate of Lactobacillus rhamnosus in Gastric fluid.

5) Alginate-Poly (ethylene glycol) Hybrid Gelispheres

A new type of hydrogel microspheres was synthesized by Redouan Mahou and Christine

Wandrey, according to them the combination of electrostatic interaction of calcium ions with

sodium alginate and the chemical reaction of vinyl sulfone-terminated poly (ethylene glycol)



368


(PEG-VS) with Threo-1,4-dimercapto-2,3-butanediol (DTT). A one-step extrusion process

under physiological conditions yielded calcium alginate-poly (ethylene glycol) hybrid

microspheres (Alg-PEG-M), an interpenetrating network with well-controllable physical

properties. It was mentioned that the permeability of the hydrogel can be tailored by

adequate choice of the arm length of PEG-VS, while the swelling degree can be tuned by

varying the PEG-VS concentration and/or by liquefaction of Calcium-alginate. It was also

given that dissolution of Calcium alginate has no significant impact on the mechanical

resistance of the obtained Poly (ethylene glycol) microspheres (PEG-M). Overall, important

physical properties of the hydrogel spheres are obtainable in the range desired for

biotechnological, biomedical, and pharmaceutical applications.

6) Multi-polyelectrolyte gelispheres

Viness Pillay, Michael P. Danckwerts statistically developed and evaluated calcium-alginate-

pectinate-cellulose acetophthalate gelisphere. Authors focus on the the complex dynamics

associated with the three key textural parameters namely matrix resilience, fracture energy,

and matrix hardness which were significantly influenced by the degree of crosslinking

achieved under various conditions of reaction.

In this technique the polymer solution for crosslinking prepared as: 1.5 g of disodium

hydrogen orthophosphate was dissolved in 80 mL of deionized water to which cellulose

acetophthalate (1.5% w/v) was added. To facilitate dissolution of cellulose acetophthalate,

the solution was magnetically stirred at 658°C, taking precautions not to introduce air

bubbles. Thereafter, sodium alginate and pectin (1.5% w/v each) was added to this solution.

This multicomponent solution was then made up to volume 100 mL with deionized water.

The crosslinking solution was prepared by dissolving 150mL of glacial acetic acid in 1000

mL of deionized water. To this acidified solution, 2% w/v calcium chloride was incorporated.

Gelispheres were formed by titration of the polymer suspension at 2 mL/min with the

crosslinking solution using flat-tip 19-guage opening. The gelispheres formed were

allowed to cure for period of 24 hour at 218°C, then the crosslinking solution decanted and

gelispheres washed and dried for 48 hour at 218°C under extractor.

7) Ionotropic gelation followed by compression

Yahya E. Choonara and colleagues developed a new method for Alginate-

Hydroxyethylcellulose Gelispheres for Controlled Intrastriatal Nicotine Release in

Parkinson’s disease. Hydroxyethylcellulose was incorporated as a reinforcing protective



369


colloidal polymer to induce interactions between the free carboxyl groups of alginate with

Hydroxy ethyl cellulose monomers. Further to prolong the release of nicotine, Gelispheres

were compressed within an external poly (lactic-co-glycolic acid) (PLGA) matrix.

CONCLUSION

The study of nanoparticle about most achieving drug delivery for antimicrobial activity

showing higher systemic effect. The Nanoparticles in the size range 1–100 nm are developing

as a class of therapeutics for cancer and antimicrobial spectrum. Nanoparticles therapeutics

can show enhanced effectiveness, while concurrently reducing side effects, owing to

properties such as more targeted localization in tumours, active cellular uptake and

antimicrobial spectrum. Application of nanoparticles in these arenas is dependent on the

ability to production particles with different chemical conformation, shape, size, and

monodispersity. In the preparation of nanoparticles are using biodegradable polymer and

advantage of bio readable polymer they shall have lesser side effect as compare to other

polymer. In the preparation methods are archiving by using different polymers and different

solvents.

REFERENCES

1. Bernabeu E, Gonzalez L, Cagel M, Gergic EP, Moretton MA, Chiappetta DA. Novel

Soluplus-TPGS mixed micelles for encapsulation of paclitaxel with enhanced in vitro

cytotoxicity on breast and ovarian cancer cell lines. Colloids and surfaces B, Biointerfaces,

2016; 140: 403-11.

2. Utku S, Ozcanhan MH, Suleyman M. Automated personnel-assets-consumables-drug

tracking in ambulance services for more effective and efficient medical emergency

interventions. Computer methods and programs in biomedicine, 2016.

3. Sherafudeen SP, Vasantha PV. Development and evaluation of in situ nasal gel

formulations of loratadine. Research in pharmaceutical sciences, 2015; 10(6): 466-76.

4. Mehanny M, Hathout RM, Geneidi AS, Mansour S. Exploring the use of nanocarrier

systems to deliver the magical molecule; Curcumin and its derivatives. Journal of

controlled release: official journal of the Controlled Release Society, 2016.

5. Kang X, Xiao HH, Song HQ, Jing XB, Yan LS, Qi RG. Advances in drug delivery system

for platinum agents based combination therapy. Cancer biology & medicine, 2015; 12(4):

362-74.



370


6. Jeon H, Kim J, Lee YM, Kim J, Choi HW, Lee J, et al. Poly-paclitaxel/cyclodextrin-SPION

nano-assembly for magnetically guided drug delivery system. Journal of controlled

release: official journal of the Controlled Release Society, 2016.

7. El-Sherbiny IM, El-Baz NM, Yacoub MH. Inhaled nano- and microparticles for drug

delivery. Global cardiology science & practice, 2015; 2015: 2.

8. Choi GH, Rhee DK, Park AR, Oh MJ, Hong S, Richardson JJ, et al. Ag

Nanoparticle/Polydopamine-Coated Inverse Opals as Highly Efficient Catalytic

Membranes. ACS applied materials & interfaces, 2016.

9. Mattos AC, Altmeyer C, Tominaga T, Khalil NM, Mainardes RM. Polymeric nanoparticles

for oral delivery of 5-fluorouracil: Formulation optimization, cytotoxicity assay and pre-

clinical pharmacokinetics study. European journal of pharmaceutical sciences: official

journal of the European Federation for Pharmaceutical Sciences, 2016.

10. Lim WK, Denton AR. Depletion-induced forces and crowding in polymer-nanoparticle

mixtures: Role of polymer shape fluctuations and penetrability. The Journal of chemical

physics, 2016; 144(2): 024904.

11. Karimi M, Ghasemi A, Sahandi Zangabad P, Rahighi R, Moosavi Basri SM, Mirshekari

H, et al. Smart micro/nanoparticles in stimulus-responsive drug/gene delivery systems.

Chemical Society reviews, 2016.

12. Holmkvist AD, Friberg A, Nilsson UJ, Schouenborg J. Hydrophobic ion pairing of a

minocycline/Ca/AOT complex for preparation of drug-loaded PLGA nanoparticles with

improved sustained release. International journal of pharmaceutics, 2016.

13. Jogala S, Rachamalla SS, Aukunuru J. Development of PEG-PLGA based intravenous low

molecular weight heparin (LMWH) nanoparticles intended to treat venous thrombosis.

Current drug delivery, 2016.

14. Jenkins SI, Weinberg D, Al-Shakli AF, Fernandes AR, Yiu HH, Telling ND, et al. 'Stealth'

nanoparticles evade neural immune cells but also evade all major brain cell populations:

Implications for PEG-based neurotherapeutics. Journal of controlled release: official

journal of the Controlled Release Society, 2016.

15. Ghasemi A, Mohtashami M, Sheijani SS, Aliakbari K. Chitosan-genipin nanohydrogel as

a vehicle for sustained delivery of alpha-1 antitrypsin. Research in pharmaceutical

sciences, 2015; 10(6): 523-34.

16. Yu C, Zhou M, Zhang X, Wei W, Chen X, Zhang X. Smart doxorubicin nanoparticles with

high drug payload for enhanced chemotherapy against drug resistance and cancer

diagnosis. Nanoscale, 2015; 7(13): 5683-90.



371


17. Wang H, Zhao Y, Wu Y, Hu YL, Nan K, Nie G, et al. Enhanced anti-tumor efficacy by co-

delivery of doxorubicin and paclitaxel with amphiphilic methoxy PEG-PLGA copolymer

nanoparticles. Biomaterials, 2011; 32(32): 8281-90.

18. Shen Y, Jin E, Zhang B, Murphy CJ, Sui M, Zhao J, et al. Prodrugs forming high drug

loading multifunctional nanocapsules for intracellular cancer drug delivery. Journal of the

American Chemical Society, 2010; 132(12): 4259-65.

19. de Sa Del Fiol F, Barberato-Filho S, de Cassia Bergamaschi C, Lopes LC, Gauthier TP.

Antibiotics and Breastfeeding. Chemotherapy, 2016; 61(3): 134-43.

20. Courtney CM, Goodman SM, McDaniel JA, Madinger NE, Chatterjee A, Nagpal P.

Photoexcited quantum dots for killing multidrug-resistant bacteria. Nature materials, 2016.

21. Andreini P, Bonechi S, Bianchini M, Garzelli A, Mecocci A. Automatic image

classification for the urinoculture screening. Computers in biology and medicine, 2016;

70: 12-22.

22. Verma A, Leader JB, Verma SS, Frase A, Wallace J, Dudek S, et al. Integrating Clinical

Laboratory Measures and Icd-9 Code Diagnoses in Phenome-Wide Association Studies.

Pacific Symposium on Biocomputing Pacific Symposium on Biocomputing, 2016; 21:

168-79.

23. Thrall JH. Moreton Lecture: Imaging in the Age of Precision Medicine. Journal of the

American College of Radiology: JACR, 2015; 12(10): 1106-11.

24. Shilovsky GA, Putyatina TS, Markov AV, Skulachev VP. Contribution of Quantitative

Methods of Estimating Mortality Dynamics to Explaining Mechanisms of Aging.

Biochemistry Biokhimiia, 2015; 80(12): 1547-59.

25. Martiny JB, Jones SE, Lennon JT, Martiny AC. Microbiomes in light of traits: A

phylogenetic perspective. Science, 2015; 350(6261): aac9323.

26. Kerr IB, Finlayson-Short L, McCutcheon LK, Beard H, Chanen AM. The 'Self' and

Borderline Personality Disorder: Conceptual and Clinical Considerations.

Psychopathology, 2015; 48(5): 339-48.

27. Dawson AM, Bettgenhaeuser J, Gardiner M, Green P, Hernandez-Pinzon I, Hubbard A, et

al. The development of quick, robust, quantitative phenotypic assays for describing the

host-nonhost landscape to stripe rust. Frontiers in plant science, 2015; 6: 876.

28. De la Calle C, Morata L, Cobos-Trigueros N, Martinez JA, Cardozo C, Mensa J, et al.

Staphylococcus aureus bacteremic pneumonia. European journal of clinical microbiology

& infectious diseases: official publication of the European Society of Clinical

Microbiology, 2016.



372


29. Zhou YF, Shi W, Yu Y, Tao MT, Xiong YQ, Sun J, et al.

Pharmacokinetic/Pharmacodynamic Correlation of Cefquinome Against Experimental

Catheter-Associated Biofilm Infection Due to Staphylococcus aureus. Frontiers in

microbiology, 2015; 6: 1513.

30. Steyn M, Buskes J. Skeletal Manifestations of TB in Modern Human Remains. Clinical

anatomy, 2016.

31. Silva-Valenzuela CA, Molina-Quiroz RC, Desai P, Valenzuela C, Porwollik S, Zhao M, et

al. Analysis of Two Complementary Single-Gene Deletion Mutant Libraries of Salmonella

Typhimurium in Intraperitoneal Infection of BALB/c Mice. Frontiers in microbiology,

2015; 6: 1455.

32. Sigurdsson S, Kristinsson KG, Erlendsdottir H, Hrafnkelsson B, Haraldsson A. Decreased

Incidence of Respiratory Infections in Children After Vaccination with Ten-valent

Pneumococcal Vaccine. The Pediatric infectious disease journal, 2015; 34(12): 1385-90.

33. Shahi SK, Kumar A. Isolation and Genetic Analysis of Multidrug Resistant Bacteria from

Diabetic Foot Ulcers. Frontiers in microbiology, 2015; 6: 1464.

34. Nam EY, Song KH, Kim NH, Kim M, Kim CJ, Lee JO, et al. Differences in characteristics

between first and breakthrough neutropenic fever after chemotherapy in patients with

hematologic disease. International journal of infectious diseases: IJID: official publication

of the International Society for Infectious Diseases, 2016.

35. Xu JH, Dai WJ, Chen B, Fan XY. Mucosal immunization with PsaA protein, using chitosan

as a delivery system, increases protection against acute otitis media and invasive infection

by Streptococcus pneumoniae. Scandinavian journal of immunology, 2015; 81(3): 177-

85.

36. Siddhapura K, Harde H, Jain S. Immunostimulatory effect of tetanus toxoid loaded

chitosan nanoparticles following microneedles assisted immunization. Nanomedicine:

nanotechnology, biology, and medicine, 2015.

37. Sher P, Oliveira SM, Borges J, Mano JF. Assembly of cell-laden hydrogel fiber into non-

liquefied and liquefied 3D spiral constructs by perfusion-based layer-by-layer technique.

Biofabrication, 2015; 7(1): 011001.

38. Shah HA, Patel RP. Statistical modeling of zaltoprofen loaded biopolymeric nanoparticles:

Characterization and anti-inflammatory activity of nanoparticles loaded gel. International

journal of pharmaceutical investigation, 2015; 5(1): 20-7.



373


39. Seelan V, Kumari HL, Kishore N, Selvamani P, Thanzami K, Pachuau L, et al. Exploitation

of novel gum Prunus cerasoides as mucoadhesive beads for a controlled-release drug

delivery. International journal of biological macromolecules, 2016.

40. 40. Sarwar A, Katas H, Samsudin SN, Zin NM. Regioselective Sequential

Modification of Chitosan via Azide-Alkyne Click Reaction: Synthesis, Characterization,

and Antimicrobial Activity of Chitosan Derivatives and Nanoparticles. PloS one, 2015;

10(4): e0123084.

41. 41. Coluccino L, Stagnaro P, Vassalli M, Scaglione S. Bioactive TGF-beta1/HA

alginate- based scaffolds for osteochondral tissue repair: design, realization and

multilevel characterization. Journal of applied biomaterials & functional materials, 2015:

0.

42. Szekalska M, Winnicka K, Czajkowska-Kosnik A, Sosnowska K, Amelian A. Evaluation

of Alginate Microspheres with Metronidazole Obtained by the Spray Drying Technique.

Acta poloniae pharmaceutica, 2015; 72(3): 569-78.

43. Rembe JD, Bohm JK, Fromm-Dornieden C, Schafer N, Maegele M, Frohlich M, et al.

Comparison of hemostatic dressings for superficial wounds using a new

spectrophotometric coagulation assay. Journal of translational medicine, 2015; 13: 375.

44. Jamshidi P, Birdi G, Williams RL, Cox SC, Grover LM. Modification of gellan gum with

nanocrystalline hydroxyapatite facilitates cell expansion and spontaneous osteogenesis.

Biotechnology and bioengineering, 2015.

45. Moxon SR, Smith AM. Controlling the rheology of gellan gum hydrogels in cell culture

conditions. International journal of biological macromolecules, 2015; 84: 79-86.

46. Hosseinnejad M, Jafari SM. Evaluation of different factors affecting antimicrobial

properties of chitosan. International journal of biological macromolecules, 2016.

47. Yao M, Zhou Y, Xue C, Ren H, Wang S, Zhu H, et al. Repair of rat sciatic nerve defect by

using allogeneic bone marrow mononuclear cells combined with chitosan/silk fibroin

scaffold. Cell transplantation, 2016.

48. Yang CH, Wang LS, Chen SY, Huang MC, Li YH, Lin YC, et al. Microfluidic assisted

synthesis of silver nanoparticle-chitosan composite microparticles for antibacterial

applications. International journal of pharmaceutics, 2016.

49. Laffleur F, Bacher L, Vanicek S, Menzel C, Muhammad I. Next generation of

buccadhesive excipient: preactivated carboxymethyl cellulose. International journal of

pharmaceutics, 2016.



374


50. Wu H, Li GN, Xie J, Li R, Chen QH, Chen JZ, et al. Resveratrol ameliorates myocardial

fibrosis by inhibiting ROS/ERK/TGF-beta/periostin pathway in STZ-induced diabetic

mice. BMC cardiovascular disorders, 2016; 16(1):5.

51. Vimalraj S, Saravanan S, Vairamani M, Gopalakrishnan C, Sastry TP, Selvamurugan N. A

Combinatorial effect of carboxymethyl cellulose based scaffold and microRNA-15b on

osteoblast differentiation. International journal of biological macromolecules, 2016.

52. Miran W, Nawaz M, Jang J, Lee DS. Conversion of orange peel waste biomass to

bioelectricity using a mediator-less microbial fuel cell. The Science of the total

environment, 2016; 547: 197-205.

53. Jain NK, Jain SK. Development and in vitro characterization of galactosylated low

molecular weight chitosan nanoparticles bearing doxorubicin. AAPS Pharm Sci Tech,

2010; 11(2): 686-97.

54. Vaezifar S, Razavi S, Golozar MA, Karbasi S, Morshed M, Kamali M. Effects of Some

Parameters on Particle Size Distribution of Chitosan Nanoparticles Prepared by Ionic

Gelation Method. Journal of Cluster Science, 2013; 24(3): 891-903.

55. Ma L, Liu C. Preparation of chitosan microspheres by ionotropic gelation under a high

voltage electrostatic field for protein delivery. Colloids and surfaces B, Biointerfaces,

2010; 75(2): 448-53.

56. Shu XZ, Zhu KJ. Chitosan/gelatin microspheres prepared by modified emulsification and

ionotropic gelation. Journal of microencapsulation, 2001; 18(2): 237-45.


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