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H O
5G PPI NH 2 + O n
H
O
5G
5GPPI
PPI HN
O H
n
O O
H
H
O O O
H - O
O H
-
n NH H N 2 2 NH2H N
2 H n
HN 2
H2N
O H2N
- H2N O
O H-
H n H
-
n
n H2N
H2N H
N N H2N
H NH2
N N O O
O N H
-
NH2
N
N
n
N NH2
O H- N N
nH N N
NH2
2 N N
H O
N -
H
O H2N N
N
N N
N
n O H
N NH2
H2N
N
NH2
O H- N N
N
n N
N
N N N NH2
N
H 2N N
-
H O
H2N
N
N NH2
n O H
N
- N N
O H N
N
H O N N NH2
N
nH N2
N
NH2
H2N N
N
H O
N -
N N N
O H
-
N
n O H
N N
H O N NH2
n H2N N N NH2
H2N N
H O
N -
N
H
- N
H2N
n O H
N
O O NH 2 N N
N
n -
H2N
H H -
H
O
n
NH2
H2N
H2N O n
O H2N
- -
H - H
H O nNH H N
2 2 H
O
H2N H2N
n
H2N
O n O
H
O O
O
H H
H
331
Purushothaman,: Synthesis and characterization of PEGylated polypropyleneimine (ppi) dendrimer
EVALUATION OF DRUG LOADED solution and replaced with the same volume of
PEGYLATED DENDRIMERS fresh PBS. The drug concentration was detected in
Morphology of the dendrimers: a spectrophotometer at 248 nm [8].
Morphology of respective drug loaded dendrimers Stability studies of drug loaded PEGylated
was observed by scanning electron microscope. A dendrimers:
small amount of nanoparticles sample has been PEGylated dendritic system loaded with
spread on a metal stub. The stub was then coated Prednisolone was exposed to conditions of
with conductive gold by Hitachi 1010 ion sputter temperature and light for 4 weeks. The
and was examined under Hitachi 3000N scanning formulation was taken in different vials and stored
electron microscope (JSM 5610 LV SEM, JEOL, in dark (amber color vials) and in light (colorless
Japan) chamber. The image was snapped at an vials) at , room temperature (40 ± 2°C) in
acceleration voltage of 20 kV with a chamber thermostatically controlled oven for a period of 4
pressure of 0.6 mmHg. weeks. The samples were analyzed every week for
Particle Size and polydispersity index any color change, drug content and drug release.
determination: The data obtained were used for the analysis of
Drug loaded dendrimers size was determined by any physical and chemical degradation, the
using a Zetasizer 300 HS (Malvern instruments required storage conditions and the precautions
UK). Samples were diluted with distilled water required for storage. The samples were initially
(2µg/ml) and measured at a temperature of 25 ºC. clear and transparent at 0 °C. The loss of drug
The diameter was calculated from the from the formulation was ascertained after storage
autocorrelation function of intensity of light conditions. The known amount of formulation was
scattered from nanoparticles. The Particles kept in benzoylated cellulose tubing (Sigma, USA)
measured are in triplicate. The polydispersity and dialyzed across the tubing. The external
index (PDI) was calculated for dispersion medium (10 ml methanol) was monitored for the
homogeneity and ranges from 0 to 1. The value content of the drugs spectrophotometrically. The
close to 0 indicated a homogeneous dispersion and percentage increase in drug release from the
greater than 0.3 indicate high heterogeneity [7]. formulation was analyzing the effects of
conditions of storage on the formulations.
Fourier transforms infrared spectroscopy (FTIR) Ex vivo studies:
and Nuclear magnetic resonance spectroscopy Hemolytic Toxicity of PEGylated dendrimer:
(NMR): The RBC suspension was obtained following the
FTIR spectra of plain dendrimer, PEGylated reported procedure for hemolytic studies. Briefly,
dendrimer, respective drug and drug loaded the RBC suspension (5% hematocrit) of the human
dendrimers were determined by using Perkin blood collected in HiAnticlot blood collection
Elmer RXI model. The pellets were prepared by vials (Himedia Labs, India). 0.5 ml of suitably
gently mixing of 1mg sample with 200mg diluted Prednisolone encapsulated, PEGylated and
potassium bromide at high compaction pressure. non-PEGylated formulations were added to 4.5 ml
The pellets thus prepared were scanned at of normal saline and incubated for 1h with RBC
resolution of 4cm-1 from 450 to 4000cm-1.The suspension [9]. Similarly, 0.5 ml of drug solution
Plain and PEGylated dendrimers were analysed by and 0.5 ml of dendrimer solution were mixed with
using Bruker DRX-300, NMR spectroscopy. The 4.5 ml of normal saline and incubated for 1h with
dendrimers were solubilised in D2O using RBC suspension. The drug and dendrimers in
methanol as co solvent and analysed at 300MHz. separate tubes were taken in such amount that the
In Vitro Release of drug from PEGylated resultant final concentrations of drug and
dendrimers: dendrimer were equivalent in all the cases. The
Drug releases from known amount of respective PEGylated system of dendrimer–drug complex
drug loaded PEGylated dendrimers were was taken in amount such that the resultant final
determined using a modified dissolution method. concentrations of drug and dendrimer were
The medium comprised of a 0.05 mol phosphate equivalent to that in non-PEGylated systems. This
buffer solution (PBS) (pH 7.4). The dialysis bags allowed comparison of the hemolysis data of the,
were filled with a known mass of plain drug and dendrimer, drug loaded dendrimers and PEGylated
drug loaded PEGylated dendritic architectures dendritic architectures to assess the effect of
(MWCO 1000 Da) separately and the dialysis bags PEGylation on hemolysis. After centrifugation,
were placed in 50 ml of PBS (pH 7.4) at 37ºC with supernatants were taken and diluted with an equal
slow magnetic stirring under sink conditions. volume of normal saline and absorbance was
Aliquots of 1 ml were withdrawn from the external measured at 540 nm. To obtain 0 and 100%
332
Purushothaman,: Synthesis and characterization of PEGylated polypropyleneimine (ppi) dendrimer
hemolysis, RBC suspension was added to 5 ml of
0.9% NaCl solution (normal saline) and 5 ml RESULTS AND DISCUSSION
distilled water, respectively. The degree of FTIR and NMR spectroscopy:
hemolytic was determined by the following PPI 5.0G dendrimers were synthesized by the
equation: procedure reported by using ethylenediamine as
initiator core. Synthesis of 0.5G PPI was
confirmed by IR peaks, mainly of nitrile at 2248
cm-1. All the nitrile terminal 0.5G PPI got
Where Abs, Abs100, and Abso are the absorbance of converted to (NH2)4, which was confirmed by IR
sample, a solution of 100% hemolysis, and a of PPI 1.0G that exhibited major peak at 3284.78
solution of 0% hemolytic; respectively. cm-1 for amine (N-H stretch). Likewise, IR peaks
Brine shrimp lethality assay: also confirmed the synthesis of PPI 5.0G
Brine shrimp lethality assay was used for dendrimers. The main peaks are of C-C bend
cytotoxic of formulations [10], according to (1115.21 cm-1); C-N stretch (1243.44 cm-1,
method of Brine shrimp (Artemia salina) nauplii 1374.50 cm-1); C-H bend (1477 cm-1); N-H
was hatched in sterile brine solution (prepared by deflection of amine (1665.40cm-1) and primary
using sea salt 38g/L and adjusted the Ph to 8.5 amine at 3410 cm-1(N-H stretch), confirming that
using 1N NaOH) under constant aeration for 38 h. nitrile terminal groups of dendrimer were
After hatching, 10 nauplii were placed in each vial converted to amine terminals. The results matched
and added 25,50,100µg/ml of prednisolone loaded with the reported synthesis of PPI dendrimers. The
PEGylated PPI dendrimers respectively in a final synthesized dendrimers were PEGylated using
volume of 5ml in each vial, maintained at 37°C for DCC and PEG 4000. IR and NMR data proved the
24 h under the light of incandescent lamp and synthesis of PEGylated dendrimers. The IR
surviving larvae were counted. Each experiment spectrum of PEGylated PPI 5.0G dendrimer
was conducted along with control (Vehicle exhibited major peak of N-H stretch of amide at
treated), as like test substances. Percentage 3324.70 cm-1. An important IR peak at 1242.75
lethality was determined by comparing the mean cm-1 of ether linkage (C-O) appears in the
surviving larvae of test and control tubes. The spectrum of PEGylated dendrimers.C-O stretch of
ED50 values were obtained using Fenny probed amide group has been found near 1624.29cm-1.
analysis software. The result for test compound The important peak of C-N stretch of amide also
was compared with the positive control appears at 2925.43 cm-1 (Figure 2 ).
Podophyllotoxine (2.5, 5, 10µg/ml)
333
Purushothaman,: Synthesis and characterization of PEGylated polypropyleneimine (ppi) dendrimer
F2 - Acquisition Parameters
Date_ 20120217
Time 16.40
INSTRUM spect
PROBHD 5 mm BBO BB-1H
PULPROG zg30
TD 65536
SOLVENT DMSO
NS 28
DS 2
SWH 8223.685 Hz
FIDRES 0.125483 Hz
AQ 3.9846387 sec
RG 101
DW 60.800 usec
DE 6.00 usec
TE 297.5 K
D1 1.00000000 sec
TD0 1
F2 - Processing parameters
SI 32768
SF 400.1299946 MHz
WDW EM
SSB 0
LB 0.30 Hz
GB 0
PC 1.00
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm
1.00
335
Purushothaman,: Synthesis and characterization of PEGylated polypropyleneimine (ppi) dendrimer
Figure 7: SEM photograph of prednisolone loaded PPI dendrimer
160
140
Particle size in (nm)
120
100
Series1
80
Series2
60
40
20
0
DLDP(1:0.5) DLDP(1:1) DLDP(1:2)
Formulation codes
time
Figure 9: Comparative dissolution profile for pure Prednisolone and prednisolone dendrimers
5G-PPI dendrimer 64
0.2 µg/ml 20.39±0.82
PEGylated-PPI dendrimer -
0.2 µg/ml 2.72 ± 1.10
DLDP(1:1) 0.2 µg/ml - 2.47± 1.02
% hemolysis produced by 5 mg/ml formulations on 5% hematocrit RBCs on incubation for 1h.
mean ±SD. (n = 3).
25
% of hemolysis
20
Series1
15
Series2
10
Series3
5
0
Plain dendrimers PEGylated DLDP(1:1) DLDP(1:1)
dendrimers
Formulation code
337
Purushothaman,: Synthesis and characterization of PEGylated polypropyleneimine (ppi) dendrimer
Figure 10: % Reduction of hemolysis after PEGylation of dendrimers
Table 4: Brine Shrimp Lethality (Cytotoxic) Assay for Drug Loaded Dendrimers
Live
Control Shrimps
ED50
S.No Test Solubility Tube Tube Tube Dose Tube Tube Tube Mean (μg/m DOF
1 2 3 µg/ml 1 2 3 l)
25 5 8 1 8 41.8 0.0015
2 DLDP DMSO 7 7 8 50 4 4 2 12
100 1 2 3 16
2.5 4 3 2 12 2.24 0.1267
3 Std DMSO 8 7 6 5 1 1 1 18
10 0 0 0 21
30
Mean survival time in
25
20
(Days)
Series1
15
Series2
10
5
0
Control Pure Prednisolone DLDP(1:1)
Treatment groups
339
Purushothaman,: Synthesis and characterization of PEGylated polypropyleneimine (ppi) dendrimer
animal model to evaluate the ability of a [14] Prasanta, PN Murthy, NK Tripathy, R
drug delivery system to promote the Panigrahi, S Behera. Investigation of
passage through the BBB, Neuroscience Drug Polymer Compatibility, journal of
Letters, 23, 12-19 (2008). Webmed Central,5, 1-20 (2012).
340