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CHAPTER 5

PREPARATION AND CHARACTERIZATION OF

PHYTOSOME NANOPARTICLES

5.1 INTRODUCTION

The 21st century has embraced the appearing of the age of

nanomedicine era and has already seen explosive growth in this

multidisciplinary field such as biomedical applications, advanced drug

carriers, new therapies, and in vivo imaging, and even possible future

applications of nanopharmaceutics to biomolecular nanotechnology. Exciting

biomedical applications for a wide variety of diseases such as cancers, human

immunodeficiency virus (HIV), fungal infections etc can be anticipated using

liposome nanomedicine. Of particular interest is the field of drug delivery, in

which recent advances in the liposome nanoparticles (LNs) have led to carrier

systems capable of encapsulating therapeutic agents ranging from

conventional drugs to the new genetic drugs (Ryo et al 2005; Fenske et al

2008; Whittenton et al 2008).

The preparation, characterization, formulation and evaluation of

liposomes and their applications as chemotherapeutic and generic drug

carriers, cosmetic and food technologies were reviewed (Torchilin 2005;

Immordino et al 2006; Wagner et al 2011; Mufamadi et al 2011). These

liposomes are composed of one or more lipid membranes surrounding discrete

aqueous compartments and can encapsulate water-soluble drugs in

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their aqueous spaces and lipid-soluble drugs within the membrane itself

(Balazs et al 2011). These vesicles release their drug contents by interacting

with cells in any one of these four ways: adsorption, endocytosis, lipid

exchange and fusion. These vesicles-entrapped drugs are distributed within

the body much differently than free drugs; when administered intravenously

to healthy animals and humans, most of the injected liposome accumulate in

the liver, spleen, lungs, bone marrow and lymph nodes (Moghimi et al 2003;

Umalkar et al 2010). Figure 5.1 depicts the schematic representation of gene

delivery using liposome. These vesicles also accumulate preferentially at the

sites of inflammation and infection and in some solid tumors; however, the

reason for this accumulation is not clear (Manjappa et al 2011). Four major

factors influencing liposomes in vivo behavior and biodistribution are as

follows; (1) tendency for liposomes to leak if cholesterol (CH) is not included

in the vesicle membrane, (2) small liposomes are cleared more slowly than

large liposomes, (3) the half-life of a liposome increases as the lipid dose

increases and (4) charged liposomes are cleared more rapidly than uncharged

systems. One concern in this use of this drug carrier in pharmaceutics is the

stability of the liposome during storage and the stability in the blood system

(Mufamadi et al 2011). Though CH increases the stability of liposomes it

unfortunately, creates certain problems when used for the treatment of

atherosclerosis, as it oxidizes, thereby creating stability problems (Samuni et

al 2000; Goyal et al 2005). These vesicles can be made in a particular size

range that makes them viable delivery vehicle through the mechanism of

digestion by phospholipase, phagocytosis by the reticuloendothelial system

and thus releasing its drug contents (Mozafari 2005; Ahsan et al 2002).

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Figure 5.1 Schematic representation of gene delivery using liposome

As an alternative method to liposome nanomedicine, a number of

researches have been performed to design and characterize the CH-free

liposome or phytosome (phyto-plant derived) drug carriers to pharmaceutics

application (Ickenstein et al 2003; Semalty et al 2010). To date, little is

known about the application of phytosome nanoparticles (PNs) for retention

of entrapped solutes and these reasons alone were sufficient to propose that

PNs may be relevant carriers for agents that are not currently retained in

conventional formulations. While in most cases natural and synthetic PLs are

the major components, different types of biocompatible plant extract can be

employed for improving the stability, encapsulation and delivering efficiency

of conventional liposome nanoparticles (LNs) (Fenske et al 2008).

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Figure 5.2 Major differences between a phytosome and a liposome

Phytosome is obtained by reacting soy PLs with the selected

botanical phytochemical derivatives in an opportune solvent. The phytosome

has more ability to carry the medicinal plant extract of hydrophilic agent

through the lipid bilayer and thus it is more bioavailable compared to

liposome. PLs from soybean (Glycine max) mainly PC is a lipophilic agent

that readily complexes polyphenolics and widely employed to make

phytosomes (Figure 5.2). Many popular standardized herbal extracts

comprising of flavanoids (FA), polyphenolics (PP), phenolic glycoside (PG),

terpenes, alkaloids and volatile oils are employed for the preparation of

phytosomes.

5.1.1 Objective of the Work

In this chapter plant extracts such as WSE and CHE used for

preparation of withania somnifera PNs (wPNs) and CH PNs (cPNs)

respectively were obtained. The prepared PNs were analyzed by DSC, TGA

and FT-IR and SEM analysis and compared with LNs.

159

Thus obtained wPNs and cPNs are expected to be used as an

injectable solution for controlled and targeted drug carrier system.

5.2 MATERIALS AND METHODS

5.2.1 Materials

All reagents and chemicals used were of analytical grade. Soya

Lecithins and cholesterol were purchased from Hi-media Ltd, India. All other

reagents and chemicals used for the study were sourced from SRL Ltd., India.

5.2.2 Preparation of PNs

The PNs were prepared by the following modified method

(Mozafari et al 2007). A known amount of PNs ingredients was added to a

preheated (60°C, 5 min) glycerol (final concentration 3%, v/v). The mixture

was further heated (60°C) while stirring (approx. 1000 rpm) on a hotplate

stirrer for a period of 45–60 min under N2 atmosphere. For the preparation of

CH-containing formulations, CH was first dissolved in the aqueous phase of

Milli Q water (autoclaved) at elevated temperatures (120°C) while stirring

(approx. 6500-24000 rpm) for a period of 15–30 min under N2 atmosphere

before adding the other components mentioned above. After preparation of

PNs samples, they were left at room temperature under N2 for 30 min to

stabilize. These PNs were produced by passing the emulsion repeatedly

through a polycarbonate membrane having pores 400 and 200 nm in diameter.

The diameters of these PNs were determined by the pore size of the

membrane used during the extrusion method. These PNs produced are

unilamellar, the residual solvent is removed by subjecting the phytosome film

to a high vacuum on a lyophilizer for 90-120 min.

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5.2.3 PSD and ZP Analyses

The PSD and ZP of LNs, wPNs and cPNs were determined by

Dynamic laser light scattering technology using a size measurer and Laser

Doppler electrophoretic mobility using the Zetasizer at 25 °C. The concentrations

of these PNs were diluted to 0.1 % (w/v) by pH 7.0 PBS (0.05 M).

5.2.4 DSC Analyses

The thermal properties of LNs, wPNs and cPNs were analyzed

using Differential Scanning Calorimetric (DSC, TA-DSC Q 200) analysis.

The thermal behaviour was studied by heating 4±0.5 mg of each individual

sample in a covered sample pan under nitrogen gas flow. The investigations

were carried out over the temperature range 25-300 °C with a heating rate of

10 °C min-1

.

5.2.5 TGA Analysis

The thermal properties of LNs, wPNs and cPNs were analyzed

using Thermal Gravimetric (TGA, TA-TGA Q 50) Analysis. The thermal

behavior was studied by heating 4±0.5 mg of each individual sample in a

sample pan under nitrogen gas flow. The investigations were carried out over

the temperature range 25-800 °C with a heating rate of 20 °C min-1

.

5.2.6 XRD Analysis

The crystalline states of LNs, wPNs and cPNs were evaluated by

XRD. Diffraction patterns were obtained on a Rigaku miniflex II desktop X-

ray diffractometer. The X-ray generator was operated at 40 KV tube voltages

and 40 mA of tube current, using the Ka lines of copper as the radiation

source. The scanning angle ranged from 1 to 60 of 45 min in step scan mode

(step width 1°/min).

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5.2.7 FT-IR Analysis

The FT-IR analyses of LNs, wPNs and cPNs were carried out using

Perkin Elmer spectrometer. The spectra represented the average of 50 scans.

All spectra were recorded from 600 to 4000 cm1 with a resolution of 4 cm

1.

KBr pellets were prepared by gently mixing 1mg sample with 100mg KBr.

5.2.8 Light Microscopic and SEM Analyses

The LNs, wPNs and cPNs were prepared for light microscopic

analysis. The PNs was diperesed in phosphate buffer saline (PBS). Finally,

the samples were observed under the light microscope.

The LNs, wPNs and cPNs were prepared for SEM. Gold coating

was performed by sputter coater. Finally, cell samples were observed under

the SEM [SEM, FEI-Quanta 200] at a beam voltage of 10 kV.

5.2.9 DPPH Radical Scavenging Activity

The capacity of wPNs and wPNs to scavenge the DPPH radical was

estimated according to the method (Siddhuraju et al 2002). To 2ml of LNs,

wPNs and cPNs, 2ml solution of DPPH 0.1mM was added separately. The

reaction mixture was shaken and incubated in the dark for 30 min, at room

temperature and the absorbance was recorded at 517 nm against methanol.

Controls containing methanol instead of the antioxidant solution and blanks

containing methanol instead of DPPH solution were also made. The

experiment was performed in triplicate. The inhibition of the DPPH radical by

the samples was calculated with reference to control absorbance. The % of

DPPH radical scavenging activity was plotted against the sample

concentration. Scavenging activity was expressed as the inhibition %

calculated using the following formula,

162

100xAbsControl

AbsSampleAbsControlactivityiradicalAnt%

5.2.10 Elution of the WSE and CHE

Determination of solubility characteristics of LNs, wPNs and cPNs

were obtained by adding excess of the samples to 5ml of water in sealed glass

container at room temperature. The liquids were shaken for 24 h and

centrifuged at 5000 rpm for 10 min. The supernatant was filtered and 1ml of

filtrate mixed with 9 ml of methanol. 1 ml of aliquot of the resulting solution

was measured at 360 nm.

5.2.11 Cell Viability Assay

Monolayers of fibroblast cell line NIH 3T3 purchased from

National Centre for Cell Science (NCCS), Pune, India, were grown on

dispersion of LNs, wPNs and cPNs on 96 well culture plate (Corning, NY)

and maintained in Dulbecco's Modified Eagles Medium (DMEM) with 10%

Fetal Calf Serum (FCS) supplemented with antibiotics (Sigma), penicillin

(120 units/ml), streptomycin (75 mg/ml), gentamycin (160 mg/ml) and

amphotericin B (3 mg/ml) at 37 °C humidified with 5 % CO2. After 24 hrs,

the MTT assay was performed to see the % cell viability.

5.2.12 Blood Compatibility Evaluation

The blood compatibility of LNs, wPNs and cPNs were evaluated

with in vitro blood perfusion approach. Equipment for continuously blood

perfusion was assembled. The systems including these PNs were rinsed with

sterilized 0.2 ml/L NaCl prior to the perfusion. 5 ml human blood (containing

5 ml/L EDTA to anti-coagulate) was circularly perfused into these PNs (the

adsorbents were previously swollen in sterilized 0.2 ml/L NaCl solution). The

163

changes in the amounts of white blood cell (WBC), red blood cell (RBC),

haemoglobin (HGB) and blood platelet (PLT) of the blood were monitored.

5.2.13 Storage Stability

The LNs, wPNs and cPNs were stored in a refrigerator at 4 °C.

Samples of 0.2 ml were taken at predetermined intervals.

5.2.14 Statistical Analysis

Results were expressed as mean values and standard deviations (±SD)

5.3 RESULTS

5.3.1 Particle Size and Zeta Potential Analyses

The analyses of the size distribution of LNs, wPNs and cPNs were

revealed by DLS. The particle sizes of the PNs were found to be in the range

of 20 to 900 nm. Almost similar trend was there in the case of LNs

(Figure 5.3). For all the nanoparticles, zeta potential between 10 to -50 mV

was observed (Figure 5.4). In contrast, the PNs sizes were strongly dependant

on the concentration, pH and sonication time in the aqueous phase during the

production process. Results of size distribution studies showed that the PNs

diameters are independent from the aqueous phase during the preparation

process. In SEM studies, the size of the PNs were observed in the range of

150-200 nm and the particles have exhibited spherical shape (Figure 5.10).

The surface charges of PNs were determined by measuring their

electrophoretic mobility. Figure 5.4 shows the zeta potential of PNs as a

function of pH ranging from pH 2 to 8 and the isoelectric point of PNs is

about 5.4. Therefore zeta potential of PNs observed 10 to -50 mV were

sufficiently high for electrostatic stabilization probably because of the charges

of phenolic glycosides in PNs.

164

0 2 4 6 8 10

0

200

400

600

800

1000

Siz

e (

nm

)

T im e (M in )

LN s

wP N s

cP N s

Figure 5.3 Particle size analyses of LNs, wPNs and cPNs

4 5 6 7 8 9 1 0

-5 0

-4 0

-3 0

-2 0

-1 0

0

1 0

2 0

Ze

ta p

ote

ntia

l (m

V)

p H

L N s

w P N s

cP N s

Figure 5.4 Zeta potential of LNs, wPNs and cPNs

10 9 8 7 6 5 4 3 2 1

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5.3.2 DSC Analysis

Figure 5.5 depicts the DSC thermograms of LNs, wPNs and cPNs.

This thermal behaviour may be ascribed to the presence of plant extract in an

amorphous form or molecularly dispersed. This effect on the crystalline habit

of WSE and CHE may be related to the preparative method of the PNs, in

which WSE and CHE may be turned from a crystalline state to an amorphous

one. It can be observed that LNs and PNs showed similar Td 46.61, 59.56 and

63.51 ºC, respectively (Table 5.1).

35 40 45 50 55 60 65 70 75

-5

-4

-3

-2

-1

0

1

Heat F

low

(W

/g)

Temperature (°C)

LNs

wPNs

cPNs

Figure 5.5 DSC analysis of LNs, wPNs and cPNs

While significant differences in main phase transition temperatures

were not observed, the enthalpy change in the PNs were reduced. This

phenomenon could be attributed to packing differences in the bilayer after

lyophilization. When the liposomes are dehydrated, the packing density of the

head groups increases, thereby increasing opportunities for van der Waals

166

interactions between the hydrocarbon chains. As a result, PNs have a higher

transition temperature and enthalpy changes than LNs. These results indicate

that the addition of WSE and CHE to LNs in some respects mimics the

addition of water, confirming the interaction between the extract and

phosphate groups. From the size and phase transition results, it can be

concluded that WSE and CHE protect the PNs membrane, but some fusion

still occurs, producing slight differences in the mean diameter, size

distribution and phase transition properties. In all these studies, the

thermogram of the complex also exhibited a single peak which was different

from the peak of phytoconstituents and PLs. This interaction may be due to

hydrophobic interaction and hydrogen bonding. The hydroxyl groups of the

phenol rings of WSE and CHE may be involved in hydrogen bonding whereas

the aromatic rings may be involved in hydrophobic interaction. As a result,

the major sharp peaks of PLs disappear and lower the phase transition

temperature. After the combination of the PLs with WSE and CHE molecule

polarity parts, the carbon–hydrogen chain in PLs could turn freely and enwrap

the PLs molecule polarity parts, which made the sequence decrease between

PLs aliphatic hydrocarbon chains.

5.3.3 TGA Analysis

The TGA analyses of LNs, wPNs and cPNs are shown in Figure

5.6. The % weight losses are presented in the Table 5.1. The TGA profile of

PNs shows an initial loss on drying below 100 °C (about 4%) and a second

weight loss between 175 °C - 250 °C due to bound water in sample. Around

80%, 72% and 70 % weight losses were observed for LNs, wPNs and cPNs

samples respectively. The results show the improved thermal stability of

wPNs and cPNs compared to LNs.

167

100 200 300 400 500 600 700

20

40

60

80

100

We

igh

t (%

)

Temperature (°C)

LNs

wPNs

cPNs

Figure 5.6 TGA analysis of LNs, wPNs and cPNs

In the above cases, PL-WSE and PL-CHE molecular interactions in

the complex lead to a radically different thermal profile and behavior of PNs

compared to LNs.

Table 5.1 DSC and TGA properties of LNs, wPNs and cPNs

Process Td (°C) % weight loss

LNs 46 3 79.75

wPNs 59 4 75.59

cPNs 63 3 72.18

5.3.4 XRD Analysis

The X-ray diffraction patterns for LNs, wPNs and cPNs are shown

in Figure 5.7. While the diffraction patterns for wPNs and cPNs show various

sharp peaks, diffraction pattern for LNs did not show any peaks in the same

region of the spectrum, indicating the crystalline and amorphous characteristic

of LNs. The appearance of PNs crystalline diffraction peaks confirmed.

168

10 20 30 40 50 60

0

200

400

600

800

1000

1200

1400

1600

1800

2000

LNs

2q [o]

Inte

nsity [A

rb.

Un

it]

0

1000

2000

3000

4000

5000

wPNs

Inte

nsity [A

rb.

Un

it]

0

1000

2000

3000

4000

5000

cPNs

Inte

nsity [

Arb

. U

nit]

Figure 5.7 XRD analysis of LNs, wPNs and cPNs

The crystallinity of wPNs and cPNs sample may be attributed to the

interaction between PLs and the WSE and CHE. The disappearance of LNs

crystalline diffraction peaks confirmed the formation of PNs. Unlike LNs,

bonding between WSE and CHE with the PLs in development of PNs, might

have resulted in the significant changes in their XRD.

169

5.3.5 FT-IR Analysis

The FT-IR spectrum of the LNs, wPNs and cPNs are shown in

Figure 5.8. The comparison of the spectral profile of the LNs and wPNs and

cPNs resulted in shifts to higher frequencies. There was an absorbance

broadening for both the C–H (CH2) asymmetric stretching peak near 2920

cm1 and the C–H (CH2) symmetric stretching peak near 2850cm

1. The

strongest bands are the CH2 stretching modes at 2920 cm1, 2850 cm

1, C=O

at 1734 cm1 and bands in the 1250 cm

1 to 1080 cm

1 region due to PO

modes of the headgroup. The choice of probe molecule in this study was

based on two criteria. The probe should be water soluble and contain a

functionality that produces a strong IR band that does not overlap with the

strong bands due to the liposomes.

1000 1500 2000 2500 3000 3500 4000

-5

0

5

10

15

20

25

30

35

40

Tra

nsm

itta

nce (

%)

Wavenumber (cm-1)

LNs

wPNs

cPNs

Figure 5.8 FT-IR analysis of LNs, wPNs and cPNs

5.3.6 Light microscopic and SEM analyses

The light microscopic images of LNs, wPNs and cPNs are shown in

Figure 5.9. The images revealed that wPNs and cPNs were uniform particles

with homogenous distribution without agglomeration.

170

LNs

wPNs

cPNs

Figure 5.9 Light micrograph analysis of LNs, wPNs and cPNs

171

LNs

wPNs

cPNs

Figure 5.10 SEM images of LNs, wPNs and cPNs

172

The SEM images of PNs are shown in Figure 5.10. SEM images of

the PNs revealed that wPNs and cPNs were spherical particles with a

homogenous distribution with smooth surfaces. The PNs existed dispersedly

in PNs system, and they were not agglomerated. In all the cases, the presence

of spherical-shaped vesicles was predominant and in most cases, they were

less than 100 nm in diameter.

5.3.7 DPPH Radical Scavenging Assay

The high DPPH radical scavenging activities of wPNs and cPNs

were more or less directly proportional to their concentration (Figure 5.11),

where as a little deviation was observed in the case of LNs. The results are

shown in Figure 5.11. The high DPPH radical scavenging activity of the

wPNs and cPNs might be due to the presence of WSE and CHE.

0 20 40 60 80 100 120 140 160

0

20

40

60

80

100

120

Rad

ica

l sca

ve

ncin

g a

ctivity (

%)

Concentration ( g)

LNs

wPNs

cPNs

Figure 5.11 DPPH radical scavenging activity of wPNs and cPNs

5.3.8 Elution of the WSE and CHE

Determination of % release of extracts of CH, WSE and CHE from

LNs, wPNs and cPNs in water are shown in Figure 5.12.

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0 2 4 6 8 10 12 14

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

% r

ele

ase

Time (h)

CH

WSE

CHE

Figure 5.12 Elution of the CH, WSE and CHE from LNs, wPNs and cPNs

The PNs showed better % release of extracts than the LNs

(Figure 5.12).Unlike the LNs (which showed a total of only 23.44% CH

release at the end of 10 h), PNs showed 71.45% and 60.42% release at the end

of 10 h of study in distilled water. PLs being amphiphilic surfactants are more

sobuble in water and hence the release of extracts increased in the PNs.

5.3.9 Cell Viability

The LNs, wPNs and cPNs exhibited 97±3 %, 98±2 % and 96±4 %

fibroblast (NIH 3T3) viability respectively (Figure 5.13) at 96 h. The high

degree of biocompatibility evidenced that PNs may be of potential use as drug

carriers. In the present studies, in vitro biocompatibility efficacy of PNs to

support cell proliferation and viability was assessed by growing NIH 3T3

fibroblast and were found to be grown uniformly with normal morphologies.

Moreover, compared with the LNs, their proliferations on PNs were

significantly regulated.

174

60

70

80

90

100

110

120

96724824

% C

ell

Via

bili

ty

Hours

LNs

wPNs

cPNs

Figure 5.13 Cell viability assays of LNs, wPNs and cPNs

5.3.10 The Blood Compatibility

In the development of LNs, wPNs and cPNs for hemoperfusion, the

blood compatibility of PNs is one of the major factors of concern. Though

blood compatibility of liposome is well known, it needs to be clarified for

these PNs. The changes in human blood contents were monitored during the

perfusion process. After the blood being cycled in native LNs for 1 h, white

blood cells (WBC), red blood cells (RBC), haemoglobins (HGB) and blood

platelets (PLT) were reduced less than 10 % as shown (Figure 5.14A-D). This

fact indicated that the native LNs exhibited blood compatibility well. The

entire PNs also show high blood compatibility. Most PLT reduction was

observed when performing blood perfusion on these PNs. Furthermore,

rinsing these PNs after perfusion, small blood clots could be observed, which

completely differed from the situation for other nanoparticles for no blood

clots could be observed. This fact ascertained the occurring of blood

coagulation on these PNs. The result might be ascribed to the existence of

PNs. Other LNs obtained showed acceptable blood compatibility.

175

0.0 0.2 0.4 0.6 0 .8 1.0

80

82

84

86

88

90

92

94

96

98

100

102

104

106

108

110

A

W B C

Rela

tive C

oun

ts/%

B lo od P erfus ion t im e /hours

LN s

w P N s

c P N s

0.0 0.2 0.4 0.6 0 .8 1.0

80

82

84

86

88

90

92

94

96

98

100

102

104

106

108

110

B

R B C

Re

lative C

ou

nts

/%

B lo od P erfus ion t im e /hours

B

C

D

0.0 0.2 0.4 0.6 0 .8 1.0

80

82

84

86

88

90

92

94

96

98

100

102

104

106

108

110

C

R G B

Rela

tive C

oun

ts/%

B lo od P erfus ion t im e /hours

L N s

w P N s

c P N s

0.0 0 .2 0 .4 0 .6 0 .8 1 .0

80

82

84

86

88

90

92

94

96

98

100

102

104

106

108

110

D

P LP

Rela

tive C

ounts

/%

B lo od P e rfus ion t im e /hou rs

LN s w P N s

cP N s

Figure 5.14 Blood compatibility of LNs, wPNs and cPNs: A) WBC, B)

RBC, C) HGB and D) PLT

176

The time courses of blood cell counts during the perfusion process

are shown (Figure 5.14a-d). The results revealed that relatively sharp change

in the blood cells occurred at the initial stage of perfusion and gradually

reached stable values. It was opposed that reduction in blood cell may result

from the cell trapping by these PNs. Once these PNs were filled with blood

cells, no more cells in the blood would be reduced.

5.3.11 Storage Stability

The PNs were subjected to storage stability study for a period of 6

months. The storage stability of LNs, wPNs and cPNs at 4 °C and at pH 7.0 is

presented in the Figure 5.15a and b.

0 1 2 3 4 5 6 7 8

40

50

60

70

80

90

100

110

120

130

A

Siz

e (

nm

)

Tim e (Day)

LNs wPNs

cPNs

0 20 40 60 80 100 120 140 160 180 200 220 240 260

40

45

50

55

60

65

70

75

80

85

90

95

100

B

Siz

e (

nm

)

T ime (Days)

LNs

wPNs

cPNs

Figure 5.15 Storage stability of LNs, wPNs and cPNs

177

It can be seen from retention ratio of LNs tended to decrease with

increasing storage period and it was decreased to 88.83% after 3 months

storage. It indicated that more plant extract leaked out from PNs with increase

of storage period. The leakage of extract from PNs might be attributed to

hydroxylation and degradation of bilayer membranes and/or vesicle

fusion/aggregation. However, PNs were stable for 3 months, during that

period, no significant differences in size distribution and mean diameter

occurred.

5.4 DISCUSSION

Development of novel drug carrier from natural resources is very

much needed because of the beneficial role of herbal drug in the management

of varied diseases. The bioavailability of lipophilic drugs when administered

orally as solid dosage forms is low. There are usually several factors

responsible for this but a particularly widespread problem is poor absorption

due to slow and incomplete drug dissolution. In this case, improved

bioavailability can be achieved by the use of carriers with medicinal plant

extract which can enhance the rate and the extent of therapeutic drug

solubilizing into aqueous intestinal fluids. PLs play a major role in drug

delivery technology. There are numerous advantages of PLs in addition to

solubilizing property while considering them for a carrier system. In the

present study, wPNs and cPNs were prepared in the presence of N2

atmosphere. The physicochemical investigations showed that WSE and CHE

formed complexes with PLs.

This work was focused on the preparation and characterization of

PNs for drug carriers, with the aim of establishing their utility for delivery of

hydrophobic drugs. Prior to establishment of these PNs formulations were

designed for drug delivery applications. There is a great deal of existing

literature on the in vitro physical and chemical properties of these PNs

178

prepared without CH, the existing literature provides a solid foundation for

the development of PNs as intravenous drug carrier systems. Despite

extensive studies of CH-free formulations, there has been little emphasis on

their application as drug carriers other than CH-rich liposome formulations

being considered as thermo sensitive formulations (Santos et al 2002 and

2004). Others have focused on the physico-chemical and biological attributes

of CH-free liposome including phase transition temperature determination by

DSC, XRD, protein binding, permeability and pharmacokinetic studies

(Ulrich et al 2003). Collectively, this study provides conclusive evidence that

these PNs have distinctive properties that may be beneficial for drug carriers.

However, when this information has been applied to drugs, the CH-free

formulation, even when stabilized by plant extracts such as PC-WSE and PC-

CHE incorporation, exhibit poor drug retention when compared to CH-

containing formulations. The use of novel PNs are an advanced dosage

formulation technology to deliver chemotherapeutics such herbal and

synthetic drugs by improved absorption and, as a result, produce better results

than those obtained by conventional herbal extracts (Huh et al 1996; Acharya

et al 2011 and Singh et al 2011; Manthena et al 2010). Water-soluble phyto-

constituent molecules (mainly WSC and CHE) can be converted into lipid-

compatible molecular complexes. These complexes are more bioavailable as

compared to simple herbal extracts owing to their enhanced capacity to cross

the lipid rich biomembranes and finally reach the circulatory system

(Bhattacharya et al 2009; Saraf, 2010; Vinod et al 2010; Sindhumol et al

2010; Kumar et al 2010; Jain et al 2010; Patela et al 2010).

This study provides, for the first time, evidence that PNs can

exhibit improved properties, thus providing the opportunity to develop such

formulations for drugs that are poorly retained in CH-containing liposomes. A

direct comparison of CH-rich LNs with other successful drug carriers

including conventional PL:CH and sterically stabilized PC-WSE and PC-CHE

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based PNs are provided. The antioxidant activities of the PNs were

significantly higher than LNs.

That the PNs could have improved pharmacokinetics and

pharmacological parameters can advantageously be used in the treatment of

liver diseases. It can also be used in anti-inflammatory agents as well as in

pharmaceutical and cosmetic compositions. PNs are obtained by reacting soy

PLs with the selected botanical derivatives such WSE and CHE. On the basis

of their physicochemical and spectroscopic characteristics, these complexes

can be considered novel entities. Likewise PNs, LNs are formed by mixing a

water soluble substance with PC in definite ratio under specific conditions.

Here, no chemical bond is formed; the PC molecules surround the water

soluble substance. There may be hundreds or even thousands of PC molecules

surrounding the water-soluble compound. The PNs process the PC with plant

components actually, form a 1:1 or a 2:1 molecular complexes depending on

the substance complexed involving chemical bonds. This difference results in

PNs being much better absorbed than LNs showing better bioavailability. PNs

have been found superior to liposomes in topical and skin care products.

PNs are a complex between a natural product and PLs, like soy

PLs. This complex is obtained by reaction of PL and the medicinal plant

extract in an appropriate solvent. PC is a bifunctional compound with the

phosphatidyl moiety being lipophilic and the choline moiety being

hydrophilic in nature. Specifically the choline head of the PC molecule binds

to these herbal phytoconstituent while the lipid soluble phosphatidyl portion

comprising the body and tail envelopes the choline bound material. Hence,

the phytoconstituents produce a lipid compatible molecular complex with

PLs, also called as phyto-PL complex. The complexes are anchored through

chemical bonds to the polar choline head of the PLs, as can be demonstrated

by specific spectroscopic techniques. Precise chemical analysis indicates that

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units of PNs are usually flavonoids, polyphenolics and phenolic glycosides

linked with at least one PC molecule. This results in a little micro to nano

sphere or cell being produced. The PNs produces a little cell, whereby the

plant extract or its active constituent could be protected from destruction

owing to the property of PC. On the basis of spectral analysis it has been

shown that the main PC-WSE and PC-CHE interaction is due to the formation

of H-bonds and hydrophobic interaction between the polar head of PLs (i.e.

phosphate and ammonium groups) and the polar functionalities of the

substrate. When treated with water, PNs assume micellar shape forming

liposomial-like structures. In liposomes the active principle is dissolved in the

floating in the layer membrane, while in PNs the active principle is anchored

to the polar head of PLs, becoming an essential and integral part of the lipid

membrane for example in the case of the PC-WSE and PC-CHE complex,

there is formation of H-bonds between the PP, FA and PG of the flavone

moiety and the phosphate ion on the PC side. The Schematic representations

of phospholipids and phenolics complexes are shown in Figure 5.16. PNs are

advanced forms of herbal products which show better absorption, utilization

and as a result will produce better results than conventional LNs, the

increased bioavailability of the PNs by pharmacokinetics and

pharmacodynamic tests in experimental animals and in human subjects.

Preparation and characterization of PLs complexes of flavonoids

for effective drug delivery have been studied (Oteiza et al 2005; Semalty et al

2010). The sterol structure-function relationships in natural and artificial

membranes have been reviewed (Bloch et al 1983). There are two important

observations pertaining to PNs that warrant further discussion. First and

foremost, flavonoids, polyphenolics and phenolic glycosides are essential

components.

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Bilayer of phospholipids

Bilayer of phospholipids with plant extract

O

OOPHO O

O

O

O-H

Phospholipids

O

OH

OH

O

H

HO

OH

O

P

O

OO

H2C

CH2

Phospholipids and phenolics complexes

Figure 5.16 Schematic representations of phospholipids and phenolics

complexes

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Consistent with CH-containing liposomes, drug loading for the

liposome is dependant on liposome composition as well as the specific

physico-chemical properties of the drug being used (Liang et al

2004).Modulation of liposomal membrane fluidity by flavonoids,

isoflavonoids and CH derivatives have been studied (Arora et al 2000; Kisoon

et al 2002). The effect of flavonoids on the PLs bilayers interaction has been

studied (Semalty et al 2010; Tedeschi et al 2010). Importantly, the CH-free

formulations may be particularly well suited for the more hydrophobic drugs.

Removing CH from the PNs may facilitate even greater flexibility and control

of drug leakage rates. The PNs technology is a breakthrough model for

marked enhancement of bioavailability, assured delivery to the tissues and no

compromise on nutrient safety.

The PNs have following advantages 1) PNs are better bioavailable

botanical extracts, dramatically enhance bioavailability due to their complex

with PLs and deliver faster and improved absorption. 2) They could enhance

the absorption of lipid insoluble polar medicinal phytochemicals through oral

as well as topical route showing better bioavailability with significantly better

therapeutic benefit 3) Dose requirement can be minimized as the

bioavailability is increased. 4) PC used in preparation of PNs could act as a

carrier and hepatoprotective substance showing the synergistic effect when

hepatoprotective substances like flavanoids are employed to form complex. 5)

PNs could be widely be used in cosmetics due to more skin penetration and

high lipid profile and 6) PNs show better stability profile owing to the

chemical bonds formed between PC molecule and phytoconstituents.

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5.5 CONCLUSIONS

PNs were successfully prepared through interactions between PL

with WSE and CHE. ZP measurement confirmed the particle sizes and

charges and the SEM analysis confirmed the surface morphology of these

PNs. It was concluded that the PLs complex with WSE and CHE might be of

potential use for improving its bioavaibility. The results showed that PLs in

PLs-WSE and CHE complex were joined by non-covalent bond and did not

form a new compound. It has been observed that the complex formed an

effective scavenger of DPPH radicals and showed strong antioxidant activity.

With the sustained release pro le, it may be possible to get a sustained action

with smaller dose. This value added herbal drug carriers system can pave the

way for large molecules to pass through the lipophilic biological membrane

and get absorbed into the systemic circulation. These PNs are supposed to

support the attachment and proliferation of cells better. These PNs based drug

carriers could be used for targeted and controlled/sustained release of drug to

lymphatic system and nervous system, blood-brain barriers and blood-

placental barriers and cellular and sub-cellular organelles etc., It would offer

several advantages including an increase in drug bioavailability and retention

at the target site and improving the adherence or adhesion to the designated

target and sustaining drug release depots. It might be effective in the

treatment of some infectious diseases such as cancer and arthritis.