1. quality of life for cancer patients. [3]

1.
INTRODUCTION

Cancer is one of the world’s most devastating
diseases, with more than 10 million new cases every year. However, mortality has
decreased in the past two years owing to better understanding of tumour biology
and improved diagnostic devices and treatments. Current cancer treatments
include surgical intervention, radiation and chemotherapeutic drugs, which often
also kill healthy cells and cause toxicity to the patient. It would therefore
be desirable to develop chemotherapeutics that can either passively or actively
target cancerous cells. Passive targeting exploits the characteristic features
of tumour biology that allow nanocarriers to accumulate in the tumour by the enhanced
permeability and retention (EPR) effect. Passively targeting nanocarriers first
reached clinical trials in the mid-1980s. 1

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The identification of appropriate targets is based
on a detailed understanding of the molecular changes underlying cancer. This
approach contrasts with the conventional, more empirical approach used to
develop cytotoxic chemotherapeutics — the main stay of cancer drug development
in past decades. 2

Current research areas include development of carriers
to allow alternative dosing routes, new therapeutic targets such as blood vessels
fueling tumor growth and targeted therapeutics that are more specific in their
activity. Clinical trials have shown that patients are open to new therapeutic
options and the goal of these new chemotherapeutics is to increase survival             time and the quality of life for
cancer patients. 3

Commonly, nanoparticles will target certain tissues
strictly because of their size and/or their physico-chemical properties; but
new types of intelligent nanoparticles that respond to an externally applied
field, be magnetic, focused heat, or light, in ways that might make them ideal
therapeutics or therapeutic delivery vehicles, are under examination. For example,
iron oxide nanoparticles, which can serve as the foundation for targeted
magnetic resonance imaging (MRI) contrast agents, can be heated to temperatures
lethal to a cancer cell merely by increasing the magnetic field at the very
location where they are bound to tumor cells. 4

Over the last few years our laboratory has been
actively involved in synthesizing biodegradable nanoparticles encapsulating
various natural products. Although the synthetic polymers display chemical stability,
their unsatisfactory biocompatibility still limits their potential clinical
applications. Because the natural polymers always show low/non toxicity, low
immunogenicity and thereafter good biocompatibility, they have been the
preferred polymers in drug delivery systems. Among the natural polymers,
alginate has become one of the most common materials used to form microcapsules.
Recently, scientists have turned their attention on tuning starch and chitosan for
use in nano-drug delivery. 5

The polymeric nanoparticles (PNPs) are prepared from
biocompatible and biodegradable polymers in size between 10- 1000 nm where the
drug is dissolved, entrapped, encapsulated or attached to a nanoparticles
matrix. PNPs are promising vehicles for drug delivery by easy manipulation to
prepare carriers with the objective of delivering the drugs to specific target;
such an advantage improves the drug safety.

The aim of this review article is to summarize about
various natural polymeric material, methods to prepare nanoparticles, mechanism
of drug release and localization of drug in target tissue, characterization of
nanoparticles and its biomedical applications.

2.
POLYMERS USED TO DESIGN NANOPARTICLES

Today, polymer nanotechnologies are an important
part of the more promising future to achieve drug delivery challenges such as
those based on drug targeting and on the delivery of undeliverable molecules
such as oligo-nucleotides or RNA interfering effectors. 6

The
table gives the various natural polymers to design nanoparticles

Polysaccharides

Starch

 

Dextran/
dextrin, cyclodextrim

 

Chitosan

 

Pectin

 

Alginate

Protein

Albumin

 

Gelatin

 

Collagen

Lipids

Phospholipids

 

Table
no. 1

In recent years, there has been an increased
interest in formulation nanoparticles (NPs) loaded selected drugs for use in
drug delivery. Particularly, polymeric NPs have obtained increasing attention
in pharmaceutics and in the fields of drug delivery. The polymeric NPs show
high effectiveness as drug delivery agents due to their specific properties,
such as extending the drug release, decreasing drug degradation, increasing
bioavailability and reducing drug toxicity. 7

Polymers obtained from natural origins have been
extensively employed not only in the food industry but also in pharmaceutical
technology. Polysaccharide polymers have emerged as being one of these because
they are less toxic, biocompatible, and biodegradable. Incorporation of the
therapeutic agent into a polymeric matrix, particularly of a natural origin,
might potentiate the protection of the biologically active compound from
degradation, control drug release, improve absorption, enhance the therapeutic
effect, and lead to the consequential decrease in the frequency of
administration. 8

Considering the potential offered by polymer
chemistry today, there are only a limited number of polymers which can be used
as constituent of nanoparticles designed to deliver drugs To explain this fact,
one should consider that a suitable polymer needs to fulfill several
requirements to be used in such an application. Firstly, it needs to be
biodegradable or at least totally eliminated from the body in a short period of
time allowing repeating administration without any risk of uncontrolled
accumulation. Secondly, it must be non toxic and non immunogenic. Its
degradation products, if any, must also be non toxic and non immunogenic.
Thirdly, it should be formulated under the form of polymer nanoparticles with
suitable properties regarding the drug delivery goal for which the nanoparticles
are designed. 9

3.
METHODS TO PREPARE POLYMERIC NANOPARTICLES

3.1
Solvent Evaporation

Solvent evaporation method first developed for preparation
of nanoparticles. In this method firstly nanoemulsion formulation prepared.
Polymer dissolved in organic solvent (dichloromethane, chloroform or ethyl
acetate). Drug is dispersed in this solution. Then this mixuture emulsified in
an aqueous phase containing surfactant (polysorbates, poloxamers sodium dodecyl
sulfates polyvinyl alcohol, gelatin) make an oil in water emulsion by using
mechanical stirring,  sonication, or
micro fluidization (high-pressure homogenization through narrow channels).
After formation of emulsion the organic solvent evaporates by increased the
temperature and reduced pressure with continuous stirring. 10, 11

                   

                                     Fig.1
Solvent Evaporation method

3.2
Dialysis

Dialysis is an effective method for preparation of
nanoparticles. In this method firstly polymer and drug dissolved in a organic
solvent. This solution added to a dialysis tube and dialysis performed against
a non-solvent miscible with the former miscible. The displacement of the
solvent inside the membrane is followed by the progressive aggregation of
polymer due to a loss of solubility and the formation of homogeneous
suspensions of nanoparticles. 12-

Fig.2 Dialysis

3.3
Supercritical fluid
technology

Supercritical fluid technology method is alternative
method because in this method organic solvents are not used which are hazardous
to the environment as well as to physiological systems. Supercritical fluids
define as a solvent at a temperature above its critical temperature at which
the fluid remains a single phase regardless of pressure. Supercritical CO2 is
the most widely used supercritical fluid because of its mild critical
conditions (Tc = 31.1 °C, Pc = 73.8 bars) it is nontoxicity, non-flammability,
and low price. 13

Mainly
supercritical fluid used in two main techniques:

1)      Supercritical
anti-solvent (SAS)

2)     
Rapid expansion of critical solution
(RESS)

Fig.3 supercritical
fluid technology

3.4
Nanoprecipitation method

This is another method which is widely used for
nanoparticle preparation which is also called solvent displacement method. This
technique was first described by Fessi at al. In this method precipitation of
polymer and drug obtained from organic solvent and the organic solvent diffused
in to the aqueous medium with or without presence of surfactant. Firstly drug
was dissolved in water, and then cosolvent (acetone used for make inner phase
more homogeneous) was added into this solution. Then another solution of
polymer (ethyl cellulose, eudragit) and propylene glycol with chloroform
prepared, and this solution was dispersed to the drug solution. This dispersion
was slowly added to 10 ml of 70% aqueous ethanol solution. After 5 minutes of
mixing, the organic solvents were removed by evaporation at 35° under normal
pressure, nanoparticles were separated by using cooling centrifuge (10000 rpm
for 20 min), supernatant were removed and nanoparticles washed with water and
dried at room temperature in a desicator. 14

               

                                      Fig.4 Nanoprecipitation method

 

3.5
Ionic Gelation Method

The polymer chitosan (Rawat S et al., 2008) was
dispersed in 50 ml of 5% glacial acetic acid solution and stirred for 4 hours
continuously then it was stabilized for overnight to obtain clear 0.4% chitosan
gel. In ionotropic gelation method (EricAllemann et al., 1993) 0.4% chitosan
gel and 0.5% of Tripolyphosphate solution (cross linking agent) were useds.
Chitosan nanoparticles formed spontaneously upon addition of 1.2 ml of an
aqueous Tripolyphosphate solution to 3 ml of chitosan solution under high speed
stirring (3000 rpm) using high speed stirrer. The resulting chitosan particle
suspensions were centrifuged at 10,000 rpm for15 minutes. The particles were
washed with distilled water and freeze dried, same method used for three
different formulations with various proportion of polymer concentration. 15

                   

Fig.5
Ionic Gelation method

 

3.6
Salting Out Method

This technique was introduced and patented by
Bindschaedler et al. and Ibrahim et al. This technique based on the separation
of water-miscible solvent from aqueous solution by salting out effect (Catarina
PR et al., 2006). In this method toxic solvents are not used. Generally acetone
is used because it is totally miscible with water and easily removed. Polymer
and drug dissolved in a solvent which emulsified into a aqueous solution
containing salting out agent (electrolytes, such as magnesium chloride and calcium
chloride, or non- electrolytes such as sucrose) but salting out can also be
produced by saturation of the aqueous phase using colloidal stabilizer/
emulsion stabilizer/ viscosity increasing agent such as polyvinyl pyrrolidone
or hydroxyl ethyl cellulose, PVA, Poly(ethylene oxide), PLGA and
poly(trimethylene carbonate). After preparation of o/w emulsion diluted with
addition of sufficient water to allow the complete diffusion of acetone into
the aqueous phase, thus inducing the formation of nanospheres. 16

           

                                      Fig.6 Salting Out method

3.7
Coacervation method

By using biodegradable hydrophilic polymers (such as
chitosan, gelatin and sodium alginate etc) nanoparticle prepared by
Coacervation method. Calvo at al prepared nanoparticles by ionic gelation
method which involves two aqeous phases. First phase contain polymer like
chitosan, a di-block co-polymer like ethylene oxide or propylene oxide
(PEO-PPO). Second phase contain polyanion sodium tripolyphosphate. Between
these two phases electrostatic interaction occurs which forms coacervates. 17

   

Fig.7
Coacervation method

 

3.8
Double Emulsification method

Emulsification and evaporation  method  have
limitation of poor entrapment of hydrophilic drugs, hence double emulsification
technique is used. Firstly w/o emulsion prepared by addition of aqeous drug
solution to organic polymer solution with continuous stirring. This prepared
emulsion another aqeous phase with vigorous stirring, resultant w/o/w emulsion
prepared.then organic solvent removed by high centrifugation. 18

Fig.8
Double Emulsification method

4.
MECHANISM OF DRUG RELEASE AND LOCALIZATION OF DRUG IN TARGET TISSUE:

There are several mechanisms which govern drug
release from polymeric nanoparticles such as: swelling of the polymer diffusion
of the adsorbed drug, drug diffusion through the polymeric matrix, polymer
erosion or degradation and a combination of both erosion and degradation as
represented in Figure 9. The initial burst release from the nanoparticles is
either because of swelling of the polymer, creating pores, or diffusion of the
drug from the surface of the polymer . Chitosan nanoparticles also exhibit a pH-dependent
drug release because of the solubility of polymer. Chitosan derivatives alter
the release of drug from the NP, affording tunable drug release  and impacting the pharmacokinetic profile of
the loaded drug.

 

Fig.9

One of example that has been proposed  about the mechanism of alginate-enclosed
chitosan-calcium phosphate-iron-saturated bovine lactoferrin nanocarrier
(AEC-CP-Fe-bLf NCs) internalization and its action inside human body., it shows
that the alginate coating of orally directed AEC-CP-Fe-bLf NCs is degraded in
the alkaline environment offered in the small intestine. Then, the alginate
coating free C-CP-Fe-bLf NCs enter the blood circulation via endocytosis and/or
transcytosis. After that, C-CP-Fe-bLf NCs are released in the tumor site by
making use of the enhanced permeability retention effect. Finally, the uptake
of C-CP-Fe-bLf NCs in to the cancer cells is based on oligosaccharide and/or
lactoferrin receptor-mediated endocytosis. 19, 20, 21

Fig.10

Both passive and active targeting have been utilized
for nanoparticle delivery. Passive targeting relies upon the unique
pharmacokinetics of nanoparticles including minimal renal clearance and
enhanced permeability and retention (EPR) through the porous angiogenic vessels
in the tumor. Surface attachment of polymers enables nanoparticles to avoid
uptake by mononuclear phagocytes in the liver, spleen, and lymph nodes, thereby
improving accumulation in the tumor. Active targeting relies on ligand directed
binding of nanoparticles to receptors expressed in the tumor. Binding of  ligands to the vasculature can occur
immediately, as it is directly accessible to nanoparticles circulating in the
blood, particles extravasate into the tissues where receptors expressed on
cancer cells and in the interstitium may be used for localization. 22, 23, 24,
25

5.
CHARACTERIZATION OF NANOPARTICLES:

5.1
In-vitro drug release study

In-vitro drug release study of CSNPs dispersion was
carried out in diffusion cell apparatus (J-FDC-07, Orchid Scientifics and
Innovations India Pvt. Ltd.) in phosphate buffer pH 6.8. At predetermined time
intervals the samples was withdrawn and replenish with fresh medium and the
absorbance was measured by HPLC (LC-2010C HT, Shimadzu, Japan) at 254 nm. Data
obtained from the in-vitro drug release for formulation in different release
medium were fitted to various kinetic models. Each experiment was performed in
triplicate. The drug release mechanism and linearization were determined by
finding the goodness of fit (R2) and sum squared of residuals (SSR) for each
kinetic model. 26

5.2
Drug Efficiency and Drug Loading

Freshly prepared NPs solution was centrifuged at
6000 rpm/min for 20 min to sediments of solid NPs. Next, the solution of drug
in the supernatant was analyzed, assuming that drug not present in the
supernatant was capsulated into PLGA NPs. The encapsulation efficiency (EE) and
the drug loading (DL) were calculated from the Equations (1) and (2).

EE(%) = Amount of drug encapsulation  × 100           (1)

Amount
of drug

DL(%) = Amount of drug (mg) in
NPs  × 100              (2)

100
mg of NPs

These results are expected due to an increase of
viscosity of organic phase. An increase in density can avoid the drug diffusion
from organic phase to the aqueous phase. Additionally, increasing of viscosity
of the solution results in an increase of the NPs size. This may be related to
an increase in amount of drugs in NPs. On the other hand, the aqueous phase
containing CPZ-HCl was buffered to pH = 9, which kept the drug from being lost.
In this pH the CPZ-HCl was insoluble in water. 27

 

5.3
Morphology of nano particles

Morphology of the NPs was investigated by scanning
electron microscopy (SEM). The NPs show spherical shape and smooth surface and
absence of different NPs size. In our study we measured the mean size diameter
by Dynamic Light Scattering (DLS) technique, which based on measuring the
Z-Average of nanoparticles. 28

 

5.4
Entrapment efficiency (%EE)

%EE of the drug was determined by High Performance
Liquid Chromatography (HPLC) (LC-2010C HT, Shimadzu, Japan). Formulations were
centrifuged at 10,000 rpm for 30 min. Supernant was collected and analyzed for
drug content at 254 nm by HPLC. The %EE was calculated as follows:

%EE
= (Sa-Sb)/Sa*100

 Where, Sa is
the total amount of drug in system, Sb is the amount of drug in supernatant
after centrifugation. 29

 

5.5
Conductivity study

The conductivity study was performed to confirm the
crosslinking reaction by conductivity meter (Systronics; 307). The change in
conductivity was measured after each mL of addition of TPP solution to the CS
solution. 30

5.6
Stability of Prepared Single and Dual Loaded NPs.

 A major area
of concern for NPs is their long-term physical and chemical stability Long-term
stability is generally defined to be at least 12 months. To advance an ARV NP
formulation towards clinical usage, the NP and incorporated ARV must be stable.
Furthermore, given the limited resources available in developing countries,
storage at room temperature is highly desirable. In this study, dual loaded NPs
stored at room temperature showed little to no change in, or zeta potential
after one year. This stability was also observed for, NPs stored at ?70?C for one year. 31

5.7 
Particle
size and Zeta potential

The size of the particles was determined by dynamic
laser scattering technique using Malvern nano S90 (Malvern Instruments, UK)
particle size analyzer at 25°C with an angle of 900. The zeta potential is
crucial parameter for stability in aqueous nanoparticulate dispersion. The zeta
potential measures the surface charge of the particles. The pellet obtained
after centrifugation of the nanoparticulate dispersion was redispersed with
water. The diluted sample was taken in a capillary type cell and the zeta
potential was determined by Zetasizer Nano ZS (Malvern Instruments, UK). 32

6.
 APPLICATIONS:

1.
One
application of nanoparticles in medicine currently being developed involved
employing nanoparticles to deliver drugs, heat, tight or other substances to
specific types of cells (such as cancer cells) particles are engineered so that
they are attracted to diseased cells, which allows direct treatment of those
cells this technique reduce damage to healthy cells in the body and allows for
earlier detection of disease. Nanotechnology offers here another challenge to
come to this goal a bit closer, to deliver the drug in the right place at right
time. 33

2.
Nanoparticles
can be used in targeted drug delivery at the site of disease to improve the
uptake of poorly soluble drugs the targeting of drugs to a specific site, and
drug bioavailability. A schematic comparison of untargeted and targeted drug
delivery systems is shown below. Several anti-cancer drugs including paclitaxel
,doxorubicin 5-fluorouracil and dexamethasone have been successfully formulated
using nanomaterials. 34

3.
A
novel approach for this technology is to use oligonucleotides for sensitizing
tumor cells to chemotherapy. The oligonucleotides are being combined with Nano
liposomes to target and deliver the nucleic acids to the cancer cells (102) and
block production of the alpha folate receptor. This block was shown to decrease
cell survival of breast cancer cell lines, and sensitized a cell line by 5-fold
to doxorubicin. This is a good example of how nanotechnologies can be used to
increase the effectiveness of existing drugs, facilitating the use of lower
dosages to decrease toxicity.

4.
Recent
research has developed number of NPs such as metal, semiconductor and polymeric
particles to be use as imaging probes, diagnosis and as delivery vehicles in
cancer therapy. NPs play an important role in cancer diagnosis. The particles
such as organic dye, doped polymer, liposome’s, and quantum dots are used in
cancer diagnosis. Multi functional NPs have the capability to simultaneously
carry therapeutic agents, squares imaging contrast agent, diamonds and
targeting moieties (circle) that can be used as anti cancer agents. Drug loaded
NPs can also be used in treating cancer in animal model. 35

5.
In
recent years, chitosan–anticancer drug conjugates have also been investigated.
Low molecular weight chitosan conjugated with paclitaxel (LMWC-PTX) was also
synthesized by chemical conjugation of LMWC and PTX through a succinate linker,
which can be cleaved at physiological conditions (E. Lee, 2008). This conjugate
was evaluated as a carrier for the oral delivery of paclitaxel. LMWC (MW80%, and
the drug loading was 0.7%.Wide?angle
X?ray diffraction
analysis showed that the entrapped paclitaxel was present in an amorphous
state, which has higher water solubility compared with the crystalline state.
Identical, rapid drug release from nanoparticles was observed in PBS and urine,
with 90% released at 37°C after 2 hours. 37, 38

 

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