INTRODUCTION:
The
success of a drug delivery system mainly depends on the rate and extent of
therapeutic agent reaching the systemic circulation. When a drug is given as a
conventional dosage form, therapeutically active concentration cannot be
maintained for an extended period of time. If an attempt is made to maintain
the therapeutic concentration by repeated administration of the active agent
results in toxic levels in the body.
Novel/
controlled drug delivery technology represents one of the broader areas of
science, which involves multidisciplinary scientific approach, contributing to
human health care.
Drug
targeting is defined as a selective drug release at specific physiological
sites, organs, tissues or cells, in which the pharmacological effect is
required. The concept of drug targeting is used in attempts to improve the
therapeutic index of drugs by increasing their localization to specific organs,
tissues or cells and by decreasing their potential toxic side effects at normal
sensitive sites.1,2
The
efficacy of many drugs is often limited by their potential to reach the site of
therapeutic action. In most cases (conventional dosage forms), only a small
amount of administered dose reaches the target site, while the majority of the
drug distributes throughout the rest of the body in accordance with its
physicochemical and biochemical properties.3
Chemotherapy
has become an integral component of cancer treatment for most cancers.
Conventional chemotherapeutic agents still exhibit poor specificity in reaching
tumour tissue and are often restricted by dose-limiting toxicity. The
combination of developing controlled release technology and targeted drug
delivery may provide a more efficient and less harmful solution to overcome the
limitations found in conventional chemotherapy. The efficacy of cancerous
chemotherapy is often limited by serious side effects because of the toxicity
of anticancer drugs to both tumour and normal cells.4,5
The development of a drug delivery system faces several
challenges: reaching the target site, which is often, far away from the
administration site (drug targeting), remaining at the target site to deliver
the drug, preferably in a time controlled manner, limiting the drug’s adverse
effects and ensuring biocompatibility. The need for intravenous (IV)
formulations and the advantage of enlarging surface contact with an external
medium to control release kinetics have encouraged the development of
nanoparticles. Despite several advancements, the drug transport at high
concentrations to solid tumours seems still to be a challenge.6
Nanoparticles have been widely attempted for delivering cancer
agents to tumours. Nanoparticles are one of the polymer-based colloidal drug
delivery systems with the size ranging from 1 nm to 1000 nm. They consist of
macromolecular materials in which the active principle (drug or biologically
active material) is dissolved, entrapped, or encapsulated, and/or to which the
active principle is adsorbed or attached. Recently, polymer nanoparticles have
been widely investigated as a carrier for drug targeting.7-10
Nanoparticles
offer an alternative delivery system for cancer therapy. These have the
potential to control the release of the drug from the formulation, improve the
drug pharmacokinetics and bio distribution, and reduce drug toxicity. Because
of their smaller size, nanoparticles with entrapped drugs may penetrate through
the tumours. The poor lymphatic drainage of tumours may result in slower
clearance of nanoparticles that accumulate in tumours. This effect is termed as
Enhanced Permeability and Retention Effect (EPR). In order to overcome the
drawbacks associated with conventional dosage forms, an attempt is being made
to develop an alternative drug delivery system in the form of nanoparticles for
achieving drug targeting in the treatment of cancer.11
MATERIALS AND METHODS:
Materials:
5-Fluorouracil
was obtained as a gift sample from Spectrochem pvt ltd; Mumbai and
biodegradable polymer bovine serum albumin was obtained from Central drug house
pvt ltd, New Delhi, India.
Preparation of Nanoparticles: Simple
Coacervation Method: 12-17
For the present
study, biodegradable polymer bovine serum albumin is used with the active
ingredient for the preparation of biodegradable nanoparticles.
5-Fluorouracil
loaded albumin nanoparticles were prepared by simple coacervation method.
Accurately weighed amount of 5-Fluorouracil was added to 2% bovine serum
albumin solution and incubated for 1 hrs. Ethanol was added carefully at a rate
of 1 ml/min from an injection under magnetic stirring. The nanoparticles so formed
were cross linked by adding 100 ml of 4% glutaraldehyde-ethanol and was stirred
continuously at room temperature for 3 hrs. The nanoparticles suspension was
then subjected to freeze drying. The dried nanoparticles obtained were then
transferred to vials.
Table 1: Formulation design of
5-Fluorouracil loaded albumin nanoparticles
|
Sl.no
|
In
gredients
|
F1
|
F2
|
F3
|
F4
|
F5
|
|
1
|
5-Fluorouracil
|
10
mg
|
20
mg
|
30
mg
|
40
mg
|
50
mg
|
|
2
|
Bovine serum
albumin
|
200
mg
|
200
mg
|
200
mg
|
200
mg
|
200
mg
|
|
3
|
Ethanol
|
20
ml
|
20
ml
|
20
ml
|
20
ml
|
20
ml
|
|
4
|
4%
Glutaraldehyde -Ethanol solution
|
100ml
|
100
ml
|
100
ml
|
100
ml
|
100
ml
|
Evaluation of Drug Loaded
Nanoparticles:
Particle Size Analysis:
Determination of
average particle size of Nanoparticles loaded with 5-Fluorouracil was carried
out by using a Malvern system, with vertically polarised light supplied by an
argon-ion laser operated at 40 mW. Experiments were carried out at a
temperature of 25 º ± 0.1º C at a measuring angle of 90º to the incident beam.18, 19
Surface
Charge Analysis:
The zeta-potential
of the nanoparticles was determined by laser Doppler anemometry. Measurements
were performed at 25 º ± 0.10º C. The nanoparticles were dispersed in 0.1 mM
NaCl solution and were taken in clear disposable zeta cell and measured.20-22
Surface
Morphology:
Scanning
electron microscopy was performed to characterize the surface morphology of the
formed nanoparticles at 20 kV. Prior to examination, samples were gold-coated
to render them electrically conductive and examined under the microscope.23
Percentage
Yield:
The measured
weight was divided by total amount of all non-volatile components which were
used for the preparation of microsphere. Percentage yield can be calculated
using the formula
% Yield = Total
weight of excipient and drug / Actual weight of product x 100
Encapsulation
Efficiency and Drug Loading:
To determine the
amount of drug encapsulated in Nanoparticles, a weighed amount (50 mg) of the
nanoparticles was suspended into 50 ml of ethanol and sonicated for 15 min in
order to extract the entrapped drug completely. The solution was filtered and 1
ml of this solution was withdrawn and diluted to 50 ml with pH 7.4 phosphate
buffer solution. This solution was assayed for drug content by UV
spectrophotometer at 266 nm. Calculating this concentration with the dilution
factor we get the percentage drug content.24-26
Encapsulation
efficiency was calculated
as
EE (%) = Actual Drug Content / Theoretical Drug Content X 100
Drug loading was calculated as
DL (%) = Actual Drug Content / Weight of Powdered Nanoparticles X 100
In-Vitro Dissolution Study:
Drug loaded
nanoparticles equivalent to 50 mg of 5-Fluorouracil was loaded into the basket
of the dissolution apparatus. Dissolution study carried out for 24 hrs in pH
7.4 phosphate buffer. 1 ml of the sample was withdrawn from the dissolution
media at suitable time intervals and diluted to 10 ml using pH 7.4 phosphate
buffer and the same amount was replaced with fresh buffer. The absorbance was
measured at 266 nm by using Shimadzu 1700 UV spectrophotometer, against a blank
solution.27-29
Stability
Study:
All the five
batches of Nanoparticles were tested for
stability studies. All the formulations were divided into 3 sample sets and
stored at 4 ± 1○C; 25± 2○C and 60 ± 5% RH; 37± 2○C and 65 ± 5% RH. After 30 days, the drug
release of selected formulations was determined by the method discussed
previously in invitro drug release.30-32
RESULTS AND DISCUSSION:
In the current
research, biodegradable macromolecular polymeric nanoparticulate drug delivery
system loaded with 5-Fluorouracil were formulated using simple coacervation
method using bovine serum albumin as biodegradable polymer. The prepared
nanoparticles are characterized for their post formulation parameters.
Particle
Size:
The mean
particle size of the nanoparticles was done by Malvern systems particle size
analyzer. With increase in drug concentration, the mean particle size of the
nanoparticles significantly increased and range was between 278.7 to 441.2 nm.
(Table 2)
Percentage
Yield:
Percentage yield
of the formulations were carried out and was found to be within the range
between 96.8 to 98.19% (Table 2).
Scanning
Electron Microscopy:
The SEM analysis
was done on the prepared nanoparticles of F5 formulation to access
their morphological and surface characteristics. Scanning electron microscopy
confirms the outer surface of F5 formulation was rough and dense,
while the internal surface was porous. (Fig 3)
Surface Charge:
The
surface charge of the optimised formulation F5 was done by the
Malvern Zetasizer and was found to be -30.3 mV which is shown in Fig 4. A high
zeta potential above 25 mW either positive or negative indicates that the
formulation is physically stable.
Fig 4: Zeta potential of Nanoparticles
loaded with 5-Fluorouracil
In-vitro Release Studies:
The In-vitro release
studies of Nanoparticles were carried out in pH 7.4 buffer as a dissolution
medium for a period of 24 hrs respectively. The release showed a biphasic
release with an initial burst effect. At the end of first 30 min drug release
was 10.15%, 17.48%, 27.01%, 25.5% and 42.92% for F1 to F5
respectively. The cumulative % release were found to be 76.12%, 82.60%, 89.95%,
95.61%, and 98.26% at the end of 24th
hrs (Table 3 and Fig 5).
Table
3: Invitro release profile of formulations F1-F5
|
Sl.
No
|
Time
(hrs)
|
Percentage Drug Release
|
|
F1
|
F2
|
F3
|
F4
|
F5
|
|
1
|
0.5
|
10.15
|
17.48
|
27.01
|
25.50
|
42.92
|
|
2
|
1
|
12.12
|
19.54
|
37.52
|
44.25
|
52.90
|
|
3
|
2
|
23.21
|
26.50
|
49.90
|
52.31
|
58.57
|
|
4
|
3
|
34.81
|
32.05
|
55.31
|
61.85
|
67.27
|
|
5
|
4
|
43.47
|
39.62
|
60.21
|
70.23
|
74.06
|
|
6
|
6
|
57.56
|
45.48
|
69.18
|
72.39
|
77.75
|
|
7
|
8
|
59.31
|
47.98
|
72.57
|
75.46
|
82.62
|
|
8
|
10
|
61.81
|
57.24
|
76.96
|
79.28
|
85.63
|
|
9
|
12
|
64.21
|
63.98
|
78.54
|
83.15
|
87.44
|
|
10
|
24
|
76.12
|
82.60
|
89.95
|
95.61
|
98.26
|
Stability
Studies:
These studies
revealed that, there is a reduction in entrapment efficiency after storage for
one month at 4 ± 1°C, 25 ± 2°C and 60 ±
5% RH and 37 ± 2°C and 65 ± 5% RH.
Formulations F1, F2, F3, F4 and F5
maintained at 4±1°C showed 63.91, 62.64, 76.12, 81.54 and 86.48 drug release
respectively after 12 hrs. There was a slight increase in drug release for
formulation maintained at 25±2°C and 60±5% RH and 37±2°C and 65±5% RH. These
results may be attributed to erosion of polymer matrix to some extent during
storage. On comparing this data with the previous release data of F1,
F2, F3, F4 and F5 it was observed
that there was no much difference in the drug release of formulation maintained
at 4±1°C.
Fig 5: Invitro dissolution profiles of prepared
formulations (F1-F5)
Table 4: Stability data of 5-Fluorouracil loaded albumin
nanoparticles of F1-F5
|
Formulation code
|
4°C± 1
|
25 ± 2°C
and 60 ± 5% RH
|
37 ± 2°C
and 65 ± 5% RH
|
|
%CDR
|
%CDR
|
%CDR
|
|
F1
|
63.91
|
69.98
|
73.84
|
|
F2
|
62.64
|
76.34
|
80.91
|
|
F3
|
76.12
|
83.76
|
85.23
|
|
F4
|
81.54
|
87.32
|
91.36
|
|
F5
|
86.48
|
90.87
|
95.15
|
CONCLUSION:
After observing all the
experimental results it was conclusively demonstrated that biodegradable
albumin nanoparticles loaded with 5-fluorouracil can be successfully formulated
by simple coacervation method. Invitro
dissolution profiles showed that the release was sustained for a period of 24
hrs. The stability studies showed that the formulations should be stored at
4±1°C. Formulation with higher concentration of drug showed optimum results
with all the evaluated parameters and hence considered as the ideal formulation.
Future research can be directed towards In-vivo
studies. This nanoparticulate technology can be further explored for the drugs
which show less half life and narrow therapeutic indices.
REFERENCES:
1.
Yokoyama M and Okano T. Adv.
Drug Delivery Rev. 1996; 21: 77-80.
2.
Mohsen J and Zahra
B. Protein nanoparticles. A unique system as drug delivery vehicles. Afr J Biotechnol. 2008; 7(25):
4926-34.
3.
Tiyaboonchai W.
Chitosan nanoparticles. A promising system for drug delivery. Naresuan Univ J. 2003; 11(3): 51-66.
4.
Gu FX, Karnik R,
Wang AZ, and Frokhzad OC. Targeted nanoparticles for cancer therapy. Nano Today. 2007; 2: 14-21.
5.
Shen Z, Wei W,
Zhao Y, and Wan J. Thermosensitive polymer-conjugated albumin nanospheres as
thermal targeting anti-cancer drug carrier. Eur. J. of Pharm. Sci. 2008; 35: 271-82.
6.
Marie G, Angelica
V, Robert G, and Florence D. Nanoparticles for drug delivery: the need for
precision in reporting particle size parameters. Eur J Pharm Biopharm. 2008; 69: 1–9.
7.
Reddy LH, Sharma
RK, Chuttani K, Mishra AK, and Murthy RSR. Influence of administration route on
tumor uptake and biodistribution of Etoposide loaded solid lipid nanoparticles
in Dalton’s lymphoma tumor bearing mice. J
Contr Release. 2005; 105: 185-98.
8.
Lang L, Ping J,
Ming C, Guoliang Z, and Fengbao Z. 5-Fluorouracil-loaded self-assembled
pH-sensitive nanoparticles as novel drug carrier for treatment of malignant
tumors. Chinese J. Chem. Eng.
2006; 14(3): 377-82.
9.
Kumar GA, Bhat A,
and Rani S. Preparation and characterization of diltiazem nanocapsules:
Influence of various polymers. Asian J
Pharm. 2010; 4(3): 224-34.
10.
Wu Y, Yang W, Wang
C, Hu J, and Fu S. Chitosan nanoparticles as a novel delivery system for
ammonium glycyrrhizinate. Int. J. of
Pharm. 2005; 295: 235-45.
11.
A. Robin, V. A. Veni,
M. Tanigaivel, J. Madhusudhanan and K. Sathish Kumar. Delivery of protein using
nanoparticles. Int J Pharm Pharm Sci. 2012; 4(4): 512-517.
12.
Das S, Banerjee R,
and Bellare J. Aspirin loaded albumin nanoparticles by coacervation.
Implications in drug delivery. Trends
Biomater. 2005; 18(2): 203-12.
13.
Battaglia
L, Gallarate M, Cavalli R, Trotta M. Solid
lipid nanoparticles produced through a coacervation method. J Microencapsul. 2010; 27(1): 78-85.
14.
L.Battaglia, I. D’
Addino, E. Peira, M. Trotta, M. Gallarate. Solid lipid nanoparticles prepared
by coacervation method as vehicles for ocular cyclosporine. J. Drug Del. Sci.
Tech. 2012; 22(2): 125-130.
15.
Hetal Thakkar, Rakesh Kumar
Sharma, Anil Kumar Mishra,
Krishna Chuttani, Rayasa Ramchandra
Murthy. Albumin microspheres
as carriers for the antiarthritic drug celecoxib. AAPS PharmSciTech. 2005; 6(1):
E65-E73.
16. Swati A.
Guhagarkar, Vinod C. Malshe, Padma V. Devarajan. Nanoparticles of Polyethylene Sebacate: A New
Biodegradable Polymer. AAPS PharmSciTech.
2009; 10(3): 935-942.
17.
Mayank D. Bhavsar, Mansoor M. Amiji. Development of Novel Biodegradable Polymeric
Nanoparticles-in-Microsphere Formulation for Local Plasmid DNA Delivery in the
Gastrointestinal Tract. AAPS PharmSciTech.2008;
9(1): 288-294.
18.
Burleson D.J,
Driessen M.D, Penn R. L. On the characterization of environmental
nanoparticles. J. Environ. Science and Health. 2004; A39 (10): 2707-2753.
19.
Englert B.C.
Nanomaterials and the environment: uses,
methods and measurement. J. of Environ.
Monit. 2007; 9: 1154-1161.
20.
Kumar PV and Jain
NK. Suppression of agglomeration of Ciprofloxacin-loaded human serum albumin
nanoparticles. AAPS PharmSciTech. 2007;
8(1): E1-E6.
21.
S.
Debnath, D. Datta, M. N. Babu, R. S. Kumar, V. Senthil. Studies on the
Preparation and Evaluation of Chitosan Nanoparticles containing Cytarabine.
International Journal of Pharmaceutical Sciences and Nanotechnology.2010; 3(2):
957-964.
22.
Kavita
Sapra and S. k. Singh. Formulation Development and Optimization of Self
Emulsifying Drug Delivery Systems of Meloxicam. Int J Pharm Pharm Sci. 2013;
5(2): 524-530.
23.
Burleson D.J,
Driessen M.D, Penn R. L. On the characterization of environmental
nanoparticles. J. Environ. Science and Health. 2004; A39 (10): 2707-2753.
24.
Amar
Singh and Amar Deep. Formulation and Evaluation of Nanoparticles containing
Losartan Potassium. International Journal of Pharmacy Research and Technology. 2011; 1(1): 17-20.
25.
Adlin
Jino Nesalin J, Gowthamarajan K and Somashekhara C.N. Formulation and
Evaluation of Nanoparticles Containing Flutamide. International Journal of
ChemTech Research. 2009; 1(4): 1331-1334.
26.
M Suganeswari, Anto Shering, K.Azhagesh
raj, P.Bharathi, B.Sathish. Preparation, Characterization and Evaluation of Nanoparticles
Containing Hypolipidemic Drug and Antihypertensive Drug. International Journal
of Pharmaceutical and Biological
Archive. 2011; 2(3): 949-53.
27.
Englert B.C.
Nanomaterials and the environment: uses,
methods and measurement. J. of Environ.
Monit. 2007; 9: 1154-1161.
28. G. Suresh, K. Manjunath, V. Venkateswarlu, V. Satyanarayana. Preparation, characterization, and invitro and invivo evaluation of lovastatin solid lipid nanoparticles. AAPS PharmSciTech. 2007;
8(1): E162-E170.
29.
S.
Tamizhrasi, A. Shukla, T. Shivkumar, V. Rathi, J. C. Rathi. Formulation and
Evaluation of Lamivudine Loaded Polymethacrylic Acid Nanoparticles.
International Journal of PharmTech Research. 2009; 1(3): 411-415
30.
Stability
Testing Guidelines: Stability testing of new drug substances and products.
London: ICH – Technical Coordination, EMEA: 2003.
31.
P. Vijayaraj Kumar, Narendr K. Jain Suppression of agglomeration of
ciprofloxacin-loaded human serum albumin nanoparticles. AAPS PharmSciTech. 2007; 8(1):
E118-E123.
32. Alia A. Badawi, Mohamed Ahmed
El-Nabarawi, Doaa Ahmed
El-Setouhy, Sami Ahmed
Alsammit. Formulation and
Stability Testing of Itraconazole Crystalline Nanoparticles. AAPS PharmSciTech. 2011;
12(3): 811-820.
.
ISSN 0975-234X (Print) www.anvpublication.org
