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Encapsulation importance in pharmaceutical area, how it is done and issues

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BT658 ASSIGNMENT 2
Encapsulation importance in pharmaceutical area,
how it is done and issues about herbal extraction.

PART 1
Importance of encapsulation in pharmaceutical area

Introduction

The advantage and benefits of immobilization technology has been applied

in various area of application, from food and beverages industry, waste and
environmental technology, plant and animal, medical and pharmaceutical, energy,
engineering and so on (1). It seems that the significance and possibility of this
technology is limitless and touches every boundary of technology out there.
Nowadays, immobilization technology and its application field has becomes so broad
and elaborate. And pharmaceutical is one area of the great interest where
immobilization has made a lot of progress to its advancement.

Immobilization has been prepared by physical or chemical modification,

however nowadays an immobilization preparation can applies the immobilization
methods of more than one type of modifications. For an example a combination of
entrapment and cross-links creates an enhanced entrapment immobilization with
additional bonding strength. Immobilization, particularly by encapsulation is a unique
method itself, as encapsulation can be combined with other methods too. However
the fundamental physics of encapsulation immobilization remains, which is it should
involves the formation of a biomaterial particle in sphere-shaped structure. The
structure then encapsulates the agents by envelope, entrap, bind, adsorb and links
or many more possible ways to immobilize an agent.
 2

An encapsulation is a vesicular immobilization system where the agents are

confined or trapped inside the vesicle of the sphere structure. The biopolymer
sphere structure can be constructed into bilayer or a single layer sphere, depends on
its properties and preparation method. Whereas, matrix-type sphere immobilization
systems do not have a hollow cavity inside its structure, instead its whole structure
are solidified and filled with the biomaterial. Bilayer sphere is made of double layer
of hydrophilic polymer layering both out and inside of the structure. It usually used
to carries agents that are hydrophilic or not interacting with the immobilizer. Single
layer of sphere has a single layer of hydrophilic biopolymer are layering outer the
structure only, the inner structure are either exposed to the other side of the
hydrophobic biopolymer providing some interaction with the agent carried. In the
sphere filled with biopolymer matrix, agents are static and trapped inside the sphere,
some matrix are diffusible to the agent.

Drugs Drugs

Immobilization Immobilization

(A) Bilayer encapsulation (B) Single layer encapsulation (C) Matrix encapsulation

Figure 1: Example of encapsulation immobilizations types

 3

Encapsulation immobilization is unique compared to the other types of

immobilizations as the encapsulation of agent provides an overall protections to the
agent from harsh environment, improves its stability, activity, targeted delivery and
sustained release. These properties especially offer huge advantages in the area of
pharmaceuticals applications. Pharmaceutical applications constantly explore new
approaches to improve drug delivery efficiency, targeted delivery, sustained activity,
extended release and to immobilize poorly soluble drugs (2). And other
immobilization methods do not provide exactly the same advantage as
encapsulation has.

Protection 

Release  Stability 
Advantage of
immobilization 

Targeting  Activity 

Diagram 1: Advantages of immobilization shown

Furthermore, an encapsulation not only envelope the agent in its inner

structure but it also allows further coating or modifications of the biomaterial’s outer
surfaces. An encapsulation on the outer surface provides an interface for
modification to improve its surface properties. Its surface property can be modified in
term of negative or positive charges, hydrophilicity, identification, recognition,
adhesion and binding to target. Surface modification has shown to greatly improve
 4

delivery of the agent. Nowadays, encapsulation immobilizations with surface

modification techniques are a very common strategy to be taken.

In this report, encapsulation immobilization importance and relevance in

pharmaceutical industry will be highlighted. In pharmaceutical application, drug
delivery is the most crucial step to determine whether a drug is effective or
ineffective (2). No matter how novel and powerful the drug it is, if it is poorly
delivered and there is no drug released at the intended target area, it will result in a
lot of loss in research and development of the drug. Whenever designing a specific
drug for a specific target, researcher should also determine its most feasible and non-
destructive delivery mechanism. Thus, successful delivery of drugs depends largely
on its drug carrier and delivery route undertaken.

1. Improved release

Prior to encapsulation, in the early days of pharmaceutical drug development

era, drugs enhancements were performed for the purpose of drug protection in the
forms of coating technology. Afterwards, roughly about 60 years ago,
microencapsulation technology for pharmaceutical drugs were discovered. The
encapsulation immobilization application mainly focusing on the controlled-release
system of drug delivery (2). Drug release cannot be controlled without coating or
encapsulation, as orally administered drug will be completely dissolved in the
instant it interacts with the solvent. Thus, drug encapsulation provides a layer of
protection for gradual release of its drug content when it is naturally dissolved,
solubilizes, diffused or erodes (3). A more stable, large-sized encapsulation has a
longer release rate of up to weeks and months.

For example, in the late 1960s vaccine drug delivery were purposed for
sustained release in blood streams from nano-capsule encapsulation system. The
sustained release of vaccine provides a more prolonged immune system stimulation
(4). Beside vaccine, sustained release were also applied to other drugs such as
 5

bioactive compound (5), protein (6), DNA (7), stem cell (8) and so on. The importance
of a sustained release of drug is, it reduces the needs for recurrent drug
administration, improved patient convenience and compliance, constant drug
stimulation and increasing drug efficacy.

Moreover, the advantage of encapsulation is the ability to control the release

of a drug remotely in the targeted reaction environment. By encapsulating a drug in a
biopolymer with a special property, drug release can be controlled when required by
fine-tuning its release system. For example, multi-vesicular liposome (MVL) is a
unique lipid encapsulation biomaterial for the purpose of controlling drug release
rate effectively. The encapsulation by MVL not only can extend the release up to
several weeks but it also can adjust the drug release rate. These two properties are
attributed to the material’s packed internal aqueous chamber, which creates a higher
mechanical strength and the presence of triglyceride as hydrophobic space filler at
the junction stabilizer, which is used to adjust its agent release rate (9).

Image 1: Transmission Electron Microscope (TEM) image of multi-vesicular liposome

encapsulated with drug (9)
 6

Encapsulation can also develop a drug release mechanism by a remote

control trigger switch. An external or internal trigger can be employed to control the
release of an encapsulated agent at a specific predetermined time. Remote release
by internal triggering of an activator to release the agent was designed in the form of
self-degrading microcapsules. Once the self-degrading microcapsule is activated, a
proteolysis enzyme will self degrade the encapsulation structure to allow release of
its agent. In the encapsulation preparation, DNA agent was encapsulated and
released at predetermined time. The use of DNA as agent was probably to avoid
auto degradation of protein agent in the encapsulation. This trigger controlled
release method were not trial on protein and other drug agent as yet. Other method
of remote switch for controlled self-degradation release system were also reported
using external physical triggers such as ultrasound, magnetic field and infrared laser.
Whereas for an internal chemical triggers, hydrolytically cleavable polyelectrolytes
and chitosanase are reported (7).

Figure 2: Encapsulation of drug with self-degradation trigger for controlled release

system of drug (7)

2. Improved delivery

With regard of pharmaceutical drug delivery route, various delivery routes can
be applied according to the required therapy, feasibility and cost. They are oral,
parenteral, transdermal and inhalation routes (10). An oral route of delivery is the
most common, preferred and widely used administration route for delivery of drugs.
However human oral route faces huge obstacles and challenging environments for
 7

the safe delivery of drugs. Drugs are exposed to removal, degradation, inactivation,
loss, and unspecific and poor adsorption issues. These are the main factors which
researchers face especially for delivery of therapeutic agents, thus immobilization by
encapsulation-overcome most the issues of oral delivery.

2.1. Gastrointestinal

In an encapsulated form, agent was more protected along the oral delivery, it
provide isolation and eliminates the contact of the sensitive and active drug agent
along the harsh environment condition of oral delivery route. For example,
antioxidant agents are known to be very sensitive to the harsh environment of
gastrointestinal tract and highly reactive with free radicals. Along the oral delivery
route especially at mouth, stomach, duodenum, jejunum and proximal ileum areas,
antioxidant are exposed to free radicals, enzymatic degradation and acidic
environment. Thus, the protection of the epigallocatechin 3-gallate, epicatechin 3-
gallate, epigallocatechin, and epicatechin in biopolymer via encapsulation has been
prepared. Encapsulation has shown to improves the agent bioavailability significantly
by avoiding free radical and enzymatic degradation (11). However in an encapsulation
system, instead of the drug agent, the encapsulation biopolymer used are exposed
to degradation from harsh environment and subsequently loss the agent carried.
However, It manages to deliver the agent at the target site successfully, although at a
lower concentration.

2.2. Blood brain barrier

Delivery of drugs into a very remote area inside human body is a very
challenging task. Blood brain barrier (BBB) remains as one of the very challenging
physical barrier for drug delivery (12). Nevertheless many diseases, which are chronic,
are related to the brain functions. Those neurological diseases such as neurological
cancer, neurovascular, neurodegenerative and so on are located and originated from
the brain areas. Neurological drugs developed and administered for oral intake
 8

become less effective due to its inability to breach the BBB and reach to the specific
neurological disease target cell. Thus, encapsulation of this neurological drug can
improve drug delivery. However, encapsulation itself only protect the drug, its
delivery was enhanced with surface modification to provided an improvement
needed to carry neurological drugs and also across BBB for delivery (13).

Improved permeability of encapsulated nanoparticle carrying drugs was

accomplished with surface modification. The first reported surface coated
encapsulated nanoparticle to show successful trans BBB transport was polysorbate
80-coated poly (butyl cyanoacrylate) (4). Polysorbate 80 (Tween 80) surface coating
was found to be a great surface coating for nanoparticle. Later on various other
encapsulation nanoparticle were prepared with polysorbate 80 (Tween 80) surface
coating. For example it has been used to coat PLGA encapsulated sphere for
nootropic agent delivery into brain (14).

It was found the BBB permeability involves a BBB membrane cell

identification of a specific surface identification to allow molecules only with this
identification to cross BBB. The surface identification involved was an apolipoprotein.
In another preparation, apolipoprotein E surface coating protein, based on the
lipoprotein receptor of BBB were utilized. It shows successful delivery of loperamide
across BBB (15).

Besides surface coating method, choosing a suitable encapsulation

biopolymer also enable enhanced BBB permeability. For example, liposome as
encapsulation biopolymer for neurological drugs such as calpain inhibitor, GABA, and
phenytoin has been reported successfully transported across BBB. Liposome
advantageous due to its biocompatible and biodegradable, however liposome
encapsulation structure suffer from poor stability and very large liposome structure
has difficulties to pass through BBB (12).
 9

Figure 3: Different types of liposome encapsulation system for trans BBB delivery of
neurological drugs (12)

3. Para-cellular permeability

Therapeutic drugs are synthesized in various shapes, sizes, molecule weight,

hydrophobicity, ionic charges, functional groups and special properties, which is
dependent on their function and purpose. These drug’s complexities present a major
obstacle for its ability to be absorbed into the target cell following a successful
delivery of an agent. On the other hand, smaller molecular size, weight, non-polar,
lipophilic molecules or cell permeable drug were more easily uptake inside the cell
via adsorption or diffusion, compared to large and complex drugs, which present
more obstacle during cell uptake (13,16). Researchers have investigated that drugs
were uptake via a para-cellular transport mechanism at cell tight junctions, especially
in the intestinal epithelium cells.

Figure 4: Varieties of drug molecule (17)

 10

(A) Aspirin (21 atoms) (B) human growth hormone (C) Ig G antibody
(21 atoms) (~3,000 atoms) (~12,000 atoms)

Figure 5: Size comparisons of various drugs

Investigations of a suitable biopolymer, which can encapsulate poorly

transported and permeate drugs for improved para-cellular transport has been
reported. There are various strategy employed in pharmaceutical area to enhance
drug permeability, such as by modifying the drug to become more permeable;
physico-chemical manipulation, complexing with enhancer, attachment of oligomers
or by modulating the cell tight junction; with chelating agents, penetration
enhancers, glucose solutions, synthetic peptide of the permeable cell and exogonus
compound influence modulation. Polymers such as poly(amidoamine), poly-D-
glutamic acid, N-trimethyl chitosan chloride, thiolated polymers and many more
were under explorations have been discovered to enhance para-cellular transport by
manipulating the structure of tight junction. These different polymer systems exhibit
different mechanism of permeability action (16).

For improved oral delivery of heparin, calcitonin and insulin drug by

enhanced para-cellular transport, encapsulation immobilization with thiolated
polycarbophil method has been reported. The encapsulation has increased the
uptake of drugs compared to free drugs solution. Besides, it also provides additional
advantage such as sustained release for up to 12 hours, enzymatic inhibition and
 11

zero side effects (18,19). The biopolymer used in the encapsulation for enhanced
para-cellular transport efficacy were attributed to its muco-adhesion property, which
can mimics naturally secreted mucus glycoprotein, providing comparable and 2
times better adhesion strength than natural muco-adhesion.

Enhancing cell permeability via encapsulation method is more advantageous

than performing structural modification of drugs. Performing encapsulation of drugs is
easier and economical rather than individually binding each drug molecule with
permeable enhancers. Moreover, drugs transported via encapsulation are in bulk,
which is higher in volume and more concentrated than releasing the drugs molecule
individually. And finally, chances that an active drugs may become deactivated or
deformed upon binding with permeable enhancer, this will effect in total loss of drug
effectiveness (16).

4. Targeting

Drugs need to be delivered to the desired target cell for it to perform specific
action. Previously, drugs were targeted to the activator according to the affinity of its
active site to the binding site of its activator or target. The results of the drug action
via affinity binding shows limited success, non-specific, costly and difficult. Drug
targeting is improved via encapsulation into particle for delivery. Liposome and
nanoparticle encapsulation and has been shown to be a good biomaterial for
targeted delivery of drugs. Targeted delivery can be designed for active or passive
targeting (3).

Examples of targeting needs for current drugs applications are as follows.

Insulin cannot be taken orally, besides its intake also needs to be specifically
delivered to inhibits hepatic glucose production (20). Therapeutic stem cell needs be
targeted to the glial tissue malignant tumor of the nervous system. There is a huge
obstacle for a new stem cell tumor therapy to deliver and target the therapeutic
stem cell to a tumor cells. Beside that, blood brain barrier and vascular dysfunction
 12

in tumor patient adding to the difficulties (8). Metformin hydrochloride, an oral anti-
diabetic drug were aimed for targeted and constant release into the upper part of
gastrointestinal tract (21).

The advantage of targeting by encapsulation, for example in the case of

insulin encapsulation strategies, is because it enabled oral drug delivery to overcome
enzymatic degradation, intestinal transport, stability issues and ability to its targeting
to hepatics cells. Besides, it also provides an alternative administration route other
than currently subcutaneous which has been the main route (20). Encapsulation of
therapeutic stem cell in synthetic extracellular matrix (sECM) also presented a new
therapeutic treatment for tumor rather than performing an invasive surgical or painful
radiotherapy. sECM-encapsulated stem cell targeting to tumor cell enables the
attachment of the encapsulate to the tumor cell, avoids being cleared and
protection from being destroyed by host immune system (8). And in another
example, metformin hydrochloride drug residence time in the stomach was
increased its while maintaining its bioavailability. Encapsulation of the agent in ethyl
cellulose microsphere shows high drug loading, in vivo study found that
encapsulation increased its bioavailability and sustained release action for more than
24 hours after oral administration route (21).

Conclusion

These are some of the examples where encapsulation immobilization is

regarded as a very important technology currently available for utilization in
pharmaceutical area. It can be summarized that encapsulation has revolutionize
pharmaceutical industry significantly. The list of the encapsulation’s importance in
pharmaceutical application in table 1 is understandably brief and incomplete at the
time being. A general review of encapsulation importance in various industry have
been reported (1,22), and the need for a comprehensive review on encapsulation’s
importance exclusively in the pharmaceutical industry is timely.
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Importance Drugs / Agent Pharmaceutical applications Reference

Controlled release Influenza virus Immunization (23)
Hypericin Active agent (5)
Tetanus toxoid Immunization (6)
DNA Genetic therapy (7)
Stem cell Stem cell therapy (8)
Triggered release Breviscapine Active agent (9)
DNA Genetic therapy (8)
Improved delivery Catechin Food additives (11)
Bacoside Neurological disorder (14)
Loperamide Neurological disorder (15)
Cellular permeability Heparin Heparin therapy (24)
Insulin Diabetic type 1 therapy (18)
Calcitonin Osteoporosis (20)
Cell Targeting Insulin Targeting to hepatic (20)
Stem Cell Targeting to tumor (8)
Metformin hydrochloride Targeting to gastrointestinal (21)

Table 1: List of encapsulation importance and pharmaceutical applications

Pharmaceutical area is always innovating and moving forward, it will adapt

new drug delivery technology or develop newer delivery technique such as drug-free
paradigm, mathematical modeling or genetic engineering (2). In the current time
being, the technology and availability of immobilization is fully embraced in its
research and development of drugs. Besides pharmaceutical area, encapsulation
immobilization technology is also an important area of application in the field of
bioprocess, enzymology, environmental, energy, and agriculture (1).
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PART 2
How encapsulation is done in pharmaceutical area

Earlier, the importance of encapsulation immobilization in pharmaceutical

area was discussed. It highlighted some example, importance and application of
encapsulation immobilization of pharmaceutical drugs. In this second part, the
encapsulation preparation method will be detailed.

Drugs Biomaterial Preparation Method Reference

Influenza virus Polymethylmethacrylate Co-polymerization (23)
Hypericin PLA, PLGA Nano-precipitation (5)
(solvent displacement)
Tetanus toxoid SB-PVAL-γ-PLGA Solvent displacement (6)
DNA CaCO3 Co-precipitation (7)
Stem cell sECM Gelation (8)
Breviscapine Multivesicular liposome Double emulsification (9)
Catechin g-PGA Polyelectrolyte (11)
Bacoside PLGA Solvent evaporation (14)
Loperamide Human serum albumin Desolvation method (15)
Heparin thiolated polycarbophil Cystine polymer (24)
Insulin PLGA Multiple emulsion (25)
Metformin hydrochloride Ethyl cellulose Emulsion solvent evaporation (20)

Table 2: Encapsulation immobilization of pharmaceutical drugs

1. Virus encapsulation by polymethylmethacrylate and polyacrylamide

Starting off with vaccine encapsulation, it is the earliest encapsulation

immobilization preparation ever reported, dated as early as 1978s. The encapsulation
was prepared via co-polymerization of polymethylmethacrylate and polyacrylamide.
The encapsulation was trialed for vaccine encapsulation using inactivated influenza
virus. The virus was encapsulated via adsorption after co-polymerization or mixed
 15

with the monomers and co-polymerized together with the encapsulation biomaterial.
Influenza immunization after encapsulation showed increased immunity than free-
vaccination (21).

2. Active agent encapsulation with PLA and PLGA

Hypericin, a natural photosensitizer is hydrophobic in nature. Thus, it is

insoluble in water and poorly adsorbed. Hypericin were accidently found on photo-
poisoned animal due to over consuming hypericin-containing plant, St. John Wort,
besides it is also can be synthetically synthesized. Its properties such as
photosensitizing, fluorescent, anti-depressant properties and side effect free, made it
a very interesting agent for photo-detection (PD) and photo-dynamic therapy (PDT)
(23).

Figure 6: Hypericin structure

Encapsulation was performed with aim to increase its solubility and delivery
as therapeutic agent. In the study, hypericin encapsulation was prepared in two
polylactic acid (PLA) polymer such as (PLA) and poly (lactic-co-glycolic) acid (PLGA)
for comparison of both polymer performances. The encapsulation was performed by
nano-precipitation method or also known as solvent displacement/diffusion/shifting
or diafiltration method. It is a patented method, which is cheap, fast, easy and
simple method. It is also mainly utilized for PLA, PLGA or other biodegradable
polyesters, which is the biomaterial of choice in this preparation. (5). PLA and PLGA
 16

are the most commonly used biopolymer due to its biodegradable, biocompatible,
synthetically produced and importantly it is FDA approved for drug application.

Figure 7: Conventional way of nano-precipitation preparation method; (A) dialysis and

(B) dropping and stirring (26)

Polymers and hypericin were dissolved together in acetone to obtain organic

phase. Then, addition of poly(vinyl alcohol) (PVAL) surfactant forces the
polymerization and sphere particulate formation of polylactic acid. Afterwards, the
solvent was removed with evaporation and un-polymerized polymer and free
hypericin were removed by centrifugation. Hypericin encapsulated PLA or PLGA were
used to deliver the agent to ovarian cancer for PD and PDT treatment. PDT perform
cancer detection and destruction when it is illuminated with appropriate light
wavelength (26).

3. Tetanus toxoid encapsulation with synthetic biomaterial

In the preparation of protein for encapsulation immobilization, various carrier

systems for protein encapsulation were reviewed. The aim of encapsulation was to
select a suitable carrier, which can withstand harsh gastrointestinal environment.
 17

Other carrier systems such as emulsions, lipid, liposomes, isocom, and polymeric
nanoparticle has been investigated and reviewed for its gastrointestinal delivery
ability, however none were suitable.

Figure 8: Overview of synthesized of SB-PVAL-γ-PLGA molecule structure (5)

Notwithstanding with currently available encapsulation biomaterial and its

shortcomings, novel biodegradable comb polyesters as candidate encapsulation
biomaterial were synthesized. The new synthetic biomaterial are known as poly(2-
sulfobutyl-vinyl alcohol)-graft-poly(d,l-lactic-co-glycolic acid) (SB-PVAL-γ-PLGA). It
provides several advantages than conventional biomaterial, such as the ability to
modify its charge and composition to better suit the needs of the drug agent. The
biomaterial was prepared into nanoparticle by solvent displacement method or also
known as nano-precipitation. And then, a model protein drug for vaccination -
tetanus toxoid, was encapsulated by adsorption (6).
 18

4. DNA encapsulation in CaCO3

DNA and Pronase® were encapsulated inside a CaCO3 spherical micro-particle

template via co-precipitation method. Calcium carbonate is a natural mineral, it is
mainly used in the construction material as cement ingredient, as calcium dietary
supplement and also as food additives. Nano-sized calcium carbonate shows special
properties such as large surface area, strength, thermal stability and suitable
immobilization. Recently, calcium carbonate nanoparticle has been used to
immobilize enzyme and proteins.

The preparation of DNA encapsulation in CaCO3 involves rapid mixing of H2O,

CaCl2, Na2CO3, Pronase and dsDNA, and then stirred on magnetic stirrer. The novelty
of this encapsulation preparation is the inclusion of Pronase® in the encapsulation of
the nanoparticle. The purpose of Pronase® here is it acts as a DNA remote release
control switch by internal trigger to lyse the encapsulation and release DNA. The
needs to lyse the whole encapsulate is ensure complete release of DNA content
from the encapsulation.

Figure 9: Schematics of DNA encapsulation in CaCO3 nanoparticle and its controlled

release mechanism (6)
 19

5. Stem cell encapsulation in synthetic extra cellular matrix

In the encapsulation preparation of therapeutic stem cell inside synthetic

Extra Cellular Matrix (sECM), a highly standardized preparation protocol was followed.
Since sECM is a commercial biomaterial product, manufacturer designs its preparation
procedure. User side preparation was quite simple and only involves a few
preparation steps such as re-suspension and crosslinking of the matrix with its
preparation reagent. Later, neural stem cell were added and allowed to gel with
sECM hydrogel in 20 minutes at room temperature. Then finally, the prepared neural
stem cell encapsulated sECM were ready to be used for therapeutic delivery.

6. Breviscapine encapsulation in multivesicular liposome

A well-known, bioactive drugs known as breviscapine is clinically used to treat

ischemic cerebrovascular and cardiovascular disease, mainly in traditional Chinese
medicine practice. The aim of the encapsulation of this drug was to extend its
circulation time inside the blood circulation, reduce administration frequency and
improve patient compliance to the drug. Previously, the drug was administered via
injection under the skin, which is slightly painful and requires sterile procedure.

Figure 10: Breviscapine molecule structure

(http://www.coompo.com/structures/Breviscapine-C4142.jpg)
 20

The researcher utilized multivesicular liposome (MVL) because of its

uniqueness in extending circulation time and its ability to adjust release rate.
Encapsulation of breviscapine inside MVL was performed via double emulsification
process to produce nanoparticle in micro-particle (NIM) encapsulation system. NIM
preparation has been prepared by various methods, such as; spray drying, intra-vitreal
formulation and supercritical fluids techniques. Double emulsification method is a
convenience and widely use method for micro-particle preparation, however reports
of its NIM preparation such as in MVL is limited (7).

Its NIM preparation method is slightly complex, mainly because it involves

double emulsification steps. The biomaterial used, which is a solution containing
various lipid compounds such as phosphatidylcholine, phosphatidylglycerol,
cholesterol and triolein or tricaprylin in chloroform diethyl ether was emulsified with
aqueous solution (first aqueous solution, containing breviscapine and sucrose in
arginine containing buffers) to obtain water-in-oil (W/O) emulsion. The emulsification
was performed by centrifugation at high speed and subsequent emulsification was
performed with glucose and L-Lysine solution, yielding water in oil in water (W/O/W)
double emulsion (second emulsion). Chloroform and diethyl ether were removed
from the second emulsion by flushing nitrogen over the surface of the mixture.

Finally the encapsulated drug is obtained after centrifugation, separated from

the solvent and non-encapsulated particles. Supernatant of the encapsulated was
washed, re-suspended in buffer solution and ready for further analysis.

7. Catechins encapsulation in γ -PGA

Catechin is a major polyphenolic compounds with various property, such as

anti-oxidant by metal chelation or free radical scavengers. Besides it is also reported
to demonstrate neuro-protection and cholesterol metabolism modulation. However
the polyphenolic compound is very sensitive to degradation and low bioavailability
 21

via oral intake. The researcher plans to protect the agent in order to avoid
degradation, improve bioavailability and enhance its delivery to gastrointestinal tract
(27).

The study has selected encapsulation in chitosan γ-PGA due to several

advantages of the biopolymer. γ-PGA is an anionic, non-toxic and edible polypeptide
found in natto, it has huge potential in food, medicine and cosmetics industry
applications. The preparation method used is a simple polyelectrolyte self-assembly
method involving an electrostatic interaction of aqueous γ-PGA added into aqueous
chitosan to build up multilayered particle with unique properties. Catechin
encapsulation was prepared by dissolving the agent with γ-PGA prior to
polyelectrolyte self-assembly. Encapsulated agent were collected in pallet after
centrifugation and re-suspended in deionized water for future analysis (11).

Figure 11: Schematic of polyelectrolyte self-assembly nanoparticle construction

(a), (c) polycations assembly, (b), (d) polyanion assemble, (e), (f) formation of shell
(11)
 22

8. Bacoside encapsulation in PLGA

Bacoside is an active agent, it has antioxidant properties which is involved in

neuro-protection of nerve cells from oxidative degradations. Thus, it has great
potential to cure Alzheimer disease aside from improving memory and enhancing
cognition. Besides, its antioxidant activity also has shown potential in other illness
such as analgesic, hepatic protection, anti-inflammation and others. Conventional
administration of the supplement is via oral delivery of free un-encapsulated agent in
tablets form. The agent delivery was suggested for improvement via encapsulation,
especially in oral delivery where it is known to involve many challenges and
exposure to harsh environment. Additionally, drug delivery to nerve cell requires
trans BBB crossing of the agent, which is an another major hurdle (28).

Figure 12: Bacoside A molecule structure

(http://www.chemicalbook.com/CAS%5CGIF%5C11028-00-5.gif)

The agent was encapsulated in PLGA nanoparticle via o/w emulsion solvent
evaporation process. It is the oldest and most commonly used method for
microencapsulation. In this method, drug substances is dispersed or dissolved in
polymer/solvent system. Then it is added to aqueous phase by continuous agitation.
This method produces hardened microsphere, which contains the drugs inside the
structure. Furthermore, the encapsulated nanoparticle was surface modified by
coating with polysorbate 80. The surface coating were performed by re-suspending
the nanoparticle in phosphate buffer saline, then the same volume of polysorbate
 23

80 was added in the buffer and incubated. Finally it will be lyophilized and
confirmed the surface coating by FTIR analysis (14).

9. Loperamide encapsulation and coating with alipoprotein E

In another example of surface modification for improved delivery, drug-

encapsulated nanoparticle surface was modified with apolipoprotein E coating. The
nanoparticles were encapsulated with loperamide as model drug for delivery, which
is poorly absorbed across BBB. The model drug was encapsulated inside human
serum albumin nanoparticle (HSA-NP) where it will be surface coated with
apolipoprotein E by covalent bonding.

Figure 13: Loperamide structure

(newdruginfo.com/pharmacopeia/usp28/v28230/uspnf/pub/images/v28230/g-489.gif)

HSA-NP were prepared according to an established method called

desolvation (14). HSA powder was initially dissolved in purified water or NaCI and
then titrated to obtain pH 7-10. Then, the solution was transformed into nanoparticle
when disolvating agent, ethanol was continuously added and stirred at room
temperature. Nanoparticles were formed and cross-linked by addition of glutahydrate
in water and continually stirred for overnight. Finally, nanoparticles were collected in
the pallet after centrifugation, then it were re-suspended in water or NaCl and
sonicated for a few minutes (15).
 24

Drugs were loaded into the nanoparticle by incubating the nanoparticles with
a solution containing drug and ethanol/water. Then, unbound or free drugs after
incubation will be removed with centrifugation and the nanoparticles were re-
suspended in water. Nanoparticle surface were modified with apolipoprotein E, the
attachment involved a strong covalent bonding. Prior to conjugation, HSA-NP was
activated with a cross-linker, NHS-PEG3400-Ma to produce a sulfhydryl-reactive
particle. And at the same time apolipoprotein E was dissolved in buffer and Traut’s
reagent, incubated and purified by size exclusion chromatography. Both activated
HSA-NP and apolipoprotein E were incubated together to conjugate them.
Conjugation of HSA-NP with apolipoprotein E was formed via heterobifunctional
cross-linking. Unreacted apolipoprotein E and HSA were centrifuged to be removed,
surface modified nanoparticle was re-dispersed in ethanol/water (29). The obtained
surface modified HAS-NP with apolipoprotein E was future analyzed.

Figure 14: Molecule structure of a surface modified HSA-NP with apolipoprotein E (15)
 25

10. Heparin encapsulation

Oral drug delivery of heparin is still the most affordable and convenience
administration method so far. However, currently administered theraputic heparin
drug were not encapsulated. Thus, encapsulation was aimed to improve its efficiency
and protection along the oral drug delivery pathway. Encapsulation can improve the
bioavailability of heparin in patient blood stream, reduce frequent hospital visits and
provides compliance and convenience to the patient.

Figure 15: Parts of heparin molecule skeletal

(http://en.wikipedia.org/wiki/File:Heparin-2D-skeletal.png)

Mucoadhesive polymers are efficient drug delivery system as it can be

targeted to muco-adhesive surfaces for a sustained drug release. Using poly(acrylic
acid)-cysteine as encapsulation carrier for heparin, significant increase of absorbed
heparin were observed compared to unmodified poly(acrylic acid). In the polymer-
cysteine preparation, initially polycarbophil was neutralized with NaOH and then
hydrated with demineralized water. The polymer’s carboxylic acid moieties was
activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Then in
pH 4-5 and L-cysteine hydrochloride was added, incubated with stirring for
conjugation with the polymer. Conjugated polymer was isolated by dialysis and
lyophilized by drying frozen aqueous polymer solution (15).
 26

11. Insulin encapsulation

Insulin is a peptide hormone composed of 51 amino acid with molecular

weight of 5808 Dalton, its origins can be traced back to billion of years ago. Various
insulin encapsulation systems have been prepared to enhance insulin oral delivery.
The encapsulation preparation methods have to consider the effect organic solvent
used in the preparation to insulin activity, exposure to harsh preparation method
may alter insulin conformation structure resulting in reduced or loss of insulin activity
(30). Polymers which has been reported to be used in insulin preparation
encapsulation such as PLA, PLGA, lipid, Eudragit, poly(methacrylic acid),
poly(ethylene glycol), hydrogel, chitosan, alginate and more under development (25).

Figure 16: Insulin molecule structure

(http://upload.wikimedia.org/wikipedia/commons/0/0d/InsulinHexamer.jpg)

Preparation methods of biopolymers to encapsulate insulin has been

performed via emulsification, internal polymerization, polymer complexation, phase
inversion, reverse micellar, sol-gel, layer by layer assembly, membrane
emulsification, coacervation and spraying. All preparation methods shows high
encapsulation efficiency, most methods are simple but only some methods such as
membrane emulsification, coacervation and spraying sees possible scaling up for
industrial production. PLGA and multiple emulsion nanoparticle preparation method
is reported as the most promising polymer for insulin encapsulation (25).
 27

12. Metformin hydrochloride encapsulation in ethyl cellulose

Metformin hydrochloride is a biguanide, an oral anti-diabetic drug for type-1

diabetes, has poor bioavailability. Metformin encapsulation was prepared by
emulsion solvent evaporation method. Biopolymer, ethyl cellulose solution was
dissolved in acetone and metformin drug was dispersed in the polymer solution with
continuous stirring. Then add the dispersion into liquid paraffin and Span 60
(continuous phase) while it was continuously stirred. Continue stirring until all solvent
removed, to obtain spherical microsphere. Microsphere was washed with petroleum
ether and air dried to improve its rigidity (21).

Figure 17: Metformin molecule structure

(http://en.wikipedia.org/wiki/File:Metformin.svg)

Conclusion

In this review, encapsulation preparation methods for pharmaceutical

application has been described for some important and interesting agents such as
virus, active agent, protein drug, DNA, stem cell, antioxidant and peptide. Any kind of
pharmaceutical drugs can be encapsulated with a suitable carrier system. PLGA are
the most common biopolymer used, its Food and Drug Administration (FDA) approval
for pharmaceutical drug encapsulation certainly has some edge.
 28

PART 3
Issues about herbal extract

Introduction

Herbal is defined as any part of the plant, which can be used for its
supplemental, medicinal, flavoring, fragrance or cooking purposes. As defined by FDA,
herbal are dietary supplemental, however medicinal herb can be developed into
drugs, following a proper drug development and evaluation protocols. More than
7000 plants have been reported to exhibit medicinal herbal properties. Nowadays,
almost 50 percent of modern prescription drugs approved by FDA are originated from
herbs or natural products (21). This is translated into more than USD 20 billion per
year worth of market share of herbal medicinal and supplements. Thus, herbal
extracts are in fact have become an important part of pharmaceutical industry.
Today there are about 2000 herbal products have been approved for sold in USA.

Historically, pharmaceutical industry can be traced back to their original root

from local apothecaries practitioners or small-scale drugs manufacturer. After more
than 100 years of pharmaceutical industry existence, herbal medicines still have a lot
meaningful impact in our daily life remedies for drugs and supplement needs. One of
the reasons of its strong acceptance in the society is because of socio-cultural
believes that traditional medicine still have an advantage over conventional drugs.
Earlier civilization has established their roots in herbal medicinal practices holistically
integrated in the whole traditional medicine system. Chinese, Indian and Nepal are
some of the main countries, which have a very long and systematic traditional
medicine practice of more than hundreds of years. World Health Organization (WHO)
estimated that about 3.4 billion people in the developing world still dependent on
plant base herbal medicine, as their primary medicinal needs (31).
 29

Herbal medicinal plants have been studied since ancient time for their
curative, treatment, ailment and preparation techniques. Nowadays, the information
of herbal plant properties still has been the on-going research to prove its efficacy,
toxicology and treatment scientifically. Most of the research out there has found that
herbal plants indeed are proven to effective to the traditional applications. Besides it
also has advantage side effects, which not only enhances treatment but also can be
applied in other ailment too.

1. Effectiveness

Herbal extract products are less regulated than pharmaceutical drugs, thus its
effectiveness sometime is questionable. There are efforts to regulate and standardize
herbal products especially by providing standardized chemical ingredient
information, associated ailment, side effects and so on (31). FDA classifies also has
herbal extract as dietary supplement, which effectively not considered as therapeutic
drugs. Thus, dietary supplement cannot be claimed it can cure a specific disease or
ailment like what drugs does.

The effectiveness of a medicinal herb extract especially for use as an

effective medicinal drug application depends on the concentration of its active agent
content (17). However some traditional drugs effect were a result of synergized
action of more than one bioactive compound. Thus, removal of other
complimentary compounds may reduce the efficacy of herbal extract healing
properties significantly (32). Elucidation of the specific compound, which exhibits
specific therapeutic effect, is the ongoing research in herbal pharmaceutical area.
Unlike drug discovery by drug engineering and synthesis, finding the active agent in
herbal extract requires isolation and purification of numerous impurities and
unknown compounds. Along downstream processing the steps of herbal extract,
compound will be loss and or deactivated, and decrease its effectiveness.
 30

A suitable herbal extraction method, which is safe, efficient, economical and

high yield, is recommended. Crude herbal extract can contain a bunch of other
unwanted impurities and a compound which is not only reduce the concentration of
the herbal active agent but also creates a bulk of mass that has no effect of action.
For use in pharmaceutical applications, herbal extract are required to be very highly
active and concentrated. However during the process of concentrating the active
agent some partial or total loss of a its active agent is expected when constituents of
an extract are isolated or purified (33).

2. Safety

Herbal medicines are known for their natural properties, it is naturally free
from toxicology elements and the active agents are mostly synthesized as plant
secondary metabolite. Generally, safety concern of herbal extract consumption is
low. Lethal or toxic dose are too low to be considered dangerous or lethal, because
herbal extract are not concentrated. However allergic reactions to herbal extract may
be attributed to the impurities such as allergens. Well-known traditional herbs has
been established and used for many generations without serious side effects. And
one of the main advantage of using herbal extract in herbal medicine mostly
reported are due to its lesser side effects compared to synthetic drugs (17).

Nevertheless, World Health Organizations (WHO) has issued Guidelines for the
Assessment of Herbal Medicine (WHO, 1996) for the quality, safety and efficacy of the
herbal medicine development. Thus any herbal medicine, which is under
development, should be supported by an appropriate study plan. The document
also especially emphasized on the clinical trials protocol development for
performing herbal medicine research, quality specification of plant materials,
preparations and guidelines.
 31

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