International Journal of Research in Pharmacy and Pharmaceutical Sciences

International Journal of Research in Pharmacy and Pharmaceutical Sciences

ISSN: 2455-698X; Impact Factor: RJIF 5.22
www.pharmacyjournal.in
Volume 1; Issue 4; September 2016; Page No. 38-45

Preformulation studies intended for targeted lamotrigine polymeric nanosuspension

1
Hussein Othman Ammar, 2 Mahmoud Mohammad Ghorab, 3 Azza Ahmed Mahmoud, 4 Iman Muhammad Higazy
1, 3, 4
Department of Pharmaceutical Technology, National Research Centre, Cairo, Egypt
1, 3
Department of Pharmaceutics and Pharmaceutical Technology, Future University, Egypt
2
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Egypt

Abstract
The purpose of this study is to screen various preformulation studies in early stages of dosage form development, in order to
reach a preliminary optimized group of lamotrigine (LTG) loaded polymeric nanoparticles, with certain characteristics that would
permit brain targeting through parenteral administration. Various preformulation studies were carried out including UV
absorbance studies, whose data were a guide to LTG solubility in various solvents, FTIR studies, where interaction between LTG
and any other preparation components was studied, investigating the appropriate cryoprotectant, “cloud point titration technique”
that indicated the adequate solvent ratio in both binary and tertiary systems, and finally transmission electron microscopy to
screen all preparations. LTG was most soluble in acetone whereas polymers had higher solubility in dichloromethane.
Pluronic®F68 was the most effective cryoprotectant producing dried re-suspendable stable formulations. Transmission electron
microscopy indicated the success of preformulation studies in achieving spherical LTG-loaded stable nano formulations whose
particle size is less than 200nm, which can pass the blood brain barrier without being stuck through the reticulo-endothelial
system.

Keywords: preformulation, brain targeting, lamotrigine, biodegradable polymer, nanoparticles

1. Introduction is a biodegradable copolymer of PLA and PCL, possessing

Preformulation involves application of biopharmaceutical good mechanical properties, and its degradation rate can be
principles to physicochemical parameters of drug substance tuned by varying the ratio of the constituent polymers [9].
with the goal of designing optimum drug delivery system in Thereby; PLCL is considered a promising biomaterial in
support of promising experimental formulations [1]. Its core pharmaceutical dosage form design and development.
objective lies in development of stable, effective and safe In this study, preformulation studies would be used to reach
dosage form, since it generates useful information to the an optimum LTG loaded PNPs formulation, intended for
formulator to design an optimum drug delivery system [2]. sustained parenteral administration. Since LTG is a CNS
Lamotrigine (LTG), is a well-tolerated anticonvulsant drug, acting drug, thereby, particle size would be the main
approved for treatment of focal and generalized epilepsy in challenge in achieving our goal, where particles should be
the elderly and in patients with epilepsies plus depressive less than 200 nm in order to pass through the blood brain
disorders [3]. It undergoes extensive metabolism, and is barrier without being subjected to the reticulo-endothelial
mainly eliminated via the kidney [4]; where 94% of the orally system.
administered dose is eliminated in urine and 2% in feces.
Unchanged LTG accounts for 10% of the products detectable 2. Materials and Methods
in urine [5]. 2.1 Materials
Polymeric nanoparticles (PNPs) have been extensively Lamotrigine was kindly donated by Delta Pharma, Egypt,
studied as promising drug delivery systems due to their Pluronic®F127, Pluronic®F68, polyvinyl alcohol (low
controlled and sustained drug release properties, subcellular molecular weight: 13000- 23000 Da), dichloromethane
size, and biocompatibility with tissue and cells, which enable (DCM) were purchased from Sigma Chemical Company, St.
them to penetrate capillaries, and be taken up by cells. Louis, USA, Polyvinyl alcohol (high molecular weight:
Thereby, increasing drug accumulation at target sites [6]. 1,15,000 Da) was purchased from Loba Chemie, Mumbai,
Inspite of the increased research work on PNPs, there India, Poly-ε-(dl-lactide-co-caprolactone); PLCL
emerges safety concerns; represented in cytotoxicity of by- (polylactide: polycaprolactone; 80: 20), IV 0.79, PLCL (25:
products and scalability. Hence, biodegradable polymers 75), IV 0.79 were purchased from Lactel Bioabsorbables,
were positioned as the safer and healthier option in USA, Ethanol absolute HPLC grade was purchased from
manufacturing PNPs [7]. Poly (dl- lactide); PLA, poly-ε- Fisher scientific, UK, Acetone for HPLC, ethylacetate
caprolactone; PCL, and their copolymers, have generated (HPLC grade Mwt. 88.10 Da),
tremendous interest because of their excellent potassium dihydrogen orthophosphate, were purchased from
biocompatibility, biodegradability, mechanical strength and Sisco Research Laboratories, Mumbai, India, Sodium
most importantly, they have been approved by the FDA for phosphate dibasic heptahydrate was purchased from Riedel-
drug delivery [8]. Poly (dl-lactide-co-ԑ-caprolactone), PLCL; de Haën, Germany.

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International Journal of Research in Pharmacy and Pharmaceutical Sciences

2.2 Methods according to the following equation: CF = Sf/Si, where, Si is

2.2.1 Assessment of calibration curves for LTG through the nanoparticle size after addition of the protectant and S f is
analytical UV studies the nanoparticle size after freeze-thawing. Protection system
Standard stock solutions of LTG were prepared and scanned was considered efficient when its CF values are lower than 2
[15]
spectrophotometrically; over the range of 200–400nm with .
double beam spectrophotometer (Shimadzu UV
spectrophotometer, 240 j/PC, Japan), against the respective 2.2.7 Lyophilization process
blank, to determine wave length of maximum absorbance The prepared nanosuspension was freeze-dried in vacuum at -
(λ max). Specificity [10] and linearity11 were assessed at 60°C and pressure of 0.3 mbar to obtain powdered NPs. Main
respective absorbance values of λmax, for each medium. drying was performed for 8.5 hr, and final drying for 30 min,
where freeze-drying was carried out in a non-aseptic area18,
2.2.2 Determination of thermodynamic solubility using Lyophilizer, FD-81, Eyela, Japan. The freeze-dried
Shake-flask method of Higuchi and Connors (1965) was samples were then stored at 8°C before analysis.
used. Samples were shaken on a rotary shaker at 37ºC until
equilibrium. The two phases are then separated by filtration 2.2.8 Optimization of PNPs by changing biodegradable
[11]
. Amount of solute in supernatant is then determined using polymer weight ratio
UV spectrophotometric analysis at the corresponding λ max of Two sub-types of PLCL; having the same intrinsic viscosity
each medium. and molecular weight, but varying poly (d,l-lactide): poly-ε-
caprolactone ratios; 80: 20 and 25: 75, respectively, were
2.2.3 Compatibility study using Fourier transform used.
infrared (FTIR)
LTG was mixed with each of the components at an 2.2.9 Optimization of PNPs by changing solvent system
appropriate ratio; equivalent to that used in formulation composition
process. Each mixture was stored in an open glass bottle at Based on solubility test, various solvent systems were used as
40°C, 75% relative humidity (RH) for one week. FTIR follows: (a) Mono solvent system: organic phase is single
spectroscopy, Shimadzu, Model 8400, Japan, was used to solvent, (b) Binary system: Cloud point titration: 125 mg
study the compatibility of pure drug and other preparation polymer was dissolved in a specified volume of organic
composites, by KBr pellet method; and scanned from 4000 to solvent, then 2 ml were poured into a 20 ml glass test tube.
400 cm-1 [12]. Resultant solution was titrated with the other organic solvent,
usually alcohol, to cloud point, where polymer precipitate.
2.2.4 Formulation of LTG polymeric nanosuspension Alcohol percentage at cloud point, CLalc, was determined as
LTG polymeric nanosuspension formulations were prepared index representing affinity between polymer and solvent 15.
by spontaneous emulsification solvent diffusion method, For surfactants; 2 ml of surfactant solution were placed in 20
where an amount of polymer weighing 125 mg was dissolved ml glass test tube and titrated with the chosen solvent
in 25 ml of organic solvent together with 25 mg of LTG mixture. The volume percentage of solvent mixture at the
(polymer: drug ratio of 5: 1). The drug-polymeric solution cloud point, CLbinary, was determined as the index
was then drop wisely poured into 50 ml of emulsifier aqueous representing affinity between surfactant and solvent mixture
[14]
phase, while agitating by a magnetic stirrer at 15,000 rpm . (c) Tertiary system: A mixture of polymer and LTG were
(Wisestir® digital hotplate magnetic stirrer MSH-30D, PMI- added into DCM-binary solvent mixture; where DCM: binary
Labortechnik Gmbh, Germany) [13]. system mixture was 1: 9 [16]. The mixture was vortexed
(Paramix II vortex, Germany) for dissolution, then slowly
2.2.5 Formula purification by solvent-evaporation method poured into aqueous surfactant solution using a high speed
The dispersion formed was transferred into a suitable bulb homogenizer (Heidolph Silent Crusher Homogeniser) at
shaped glass flask to be condensed to 25 ml through 24,000 rpm for 5 min or magnetic stirrer at speed 15000 for
evaporation under reduced pressure (Rotavapor, Heidolph 10 min.
VV 2000, Germany), at 60°C for about 30 min. It was then
ultra-centrifuged at 24,000 rpm for 30 min at -4°C, and the 2.2.10 Optimization of PNPs by changing surfactant type
precipitate was then washed with 25 ml phosphate buffer (pH and concentration
7.4). This process was repeated three times to remove any Four emulsifiers were used: HPVA (13,000-23,000 Da), and
residuals [14]. LPVA (115,000 Da), Pluronic®F68 and Pluronic®F127.
Various concentrations of emulsifiers were used: 0.5, 1, 2 and
2.2.6 Freeze-thaw test for selection of suitable 4 %w/v for PVAs, and 1.25 and 2.5%w/v Pluronics ®, and
cryoprotectant examined while keeping aqueous: organic phase ratio
Nanoparticle dispersion was freezed for 12 h. Exactly 5 ml of constant (2: 1), along all experiments.
surfactant were added drop-wisely to the finally LTG loaded
formulation, to be then homogenized on magnetic stirrer for 2.2.11 Transmission electron microscopy (TEM)
less than 1 min at 500 rpm, and subsequently poured into a Morphology of various PNPs were examined by TEM;
petri dish and put into freezer overnight, followed by thawing operated at 120 kV (JEOL-JEM-1230, Japan) using computer
at 28ºC. Size and size distribution before and after freeze– program named AMT Image Capture Engine V601, USA.
thawing were measured by Zetasizer Nano ZS (Malvern Particles were stained with phosphotungestic acid (2%w/v)
Instruments, UK). Cryoprotection factor (CF) was calculated and placed on copper grids with Formvar films for viewing.

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International Journal of Research in Pharmacy and Pharmaceutical Sciences

3. Results coefficient of determination (R2) was calculated, as reference

3.1 Assessment of calibration curves for LTG through to degree of linearity; tested by plotting data and looking for
analytical UV studies curvature, indicating a positive correlation showing an
Various standard curves of LTG were assessed at intercept in the regression model [17]. However, PLCL (80:
corresponding λmax, listed in table (1), and method specificity 20) and (25: 75), did not show any λ max when preparing their
was demonstrated through standard deviation, where λ max for standard solutions in variable solvents.
each solvent, proved to be specific, (p≥0.05). In addition;
Table 1: Maximum absorbance wavelength (λmax) and linearity factor values for LTG
Organic solvents Maximum absorbance wavelength (λmax ± SD) R2 ± SD |r|
Acetone 333.4 ± 0.01 0.997 ± 0.002 0.998
Ethanol 306.2 ± 0.03 0.998 ± 0.001 0.999
Methanol 274.6 ± 0.13 0.992 ± 0.004 0.996
Acetonitrile 299.2 ± 0.10 0.998 ± 0.001 0.999
DCM 268.4 ± 0.16 0.992 ± 0.003 0.996
Phosphate buffer pH 7.4 306.5 ± 0.01 0.999 ± 0.001 0.999

3.2 Determination of thermodynamic solubility factor for qualitative solubility of polymer. Results shown in
Minimum required solubility (MRS), is used to evaluate table (2), revealed that both polymers were highly soluble in
solubility [18] of LTG, PLCL (80: 20) and (25: 75). Both; time DCM > acetone > acetonitrile > ethanol > methanol. Results
and amount required for polymer solution to reach saturation also indicate that LTG was most soluble in acetone >
against a fixed amount of solvent, were used as comparative methanol > ethanol > acetonitrile > DCM.
Table 2. Solubility data of biodegradable polymers and LTG in various solvents
Time for saturation (hr ± SD) Mean MRS (mg/ml ± SD)
Solvent
PLCL (80: 20) PLCL (25: 75) PLCL (80: 20) PLCL (25: 75) LTG
Ethanol 24 ± 6.5 24 ± 3.50 5.0 ± 0.82 7.5 ± 1.04 32.51 ± 4.08
Acetone 36 ± 4.3 36 ± 5.25 25.0 ± 3.33 22.5 ± 2.97 117.09 ± 20.71
Acetonitrile 3 ± 0.5 3 ± 0.25 15.0 ± 2.98 17.5 ± 2.15 18.77 ± 2.77
Methanol 6 ± 1.2 6 ± 1.50 2.5 ± 0.20 2.5 ± 0.01 85.05 ± 11.40
DCM 72 ± 8.5 72 ± 6.00 50.0 ± 6.13 60.0 ± 4.71 8.40 ± 0.30

3.3 Compatibility study using Fourier transform infrared molecular size and partially amorphous nature [22]. FTIR
(FTIR) spectra of PLCL (80: 20) and (25: 75), were all compatible
All readings obtained are within ranges reported in previous with data reported in literature [23]. Randomly chosen physical
studies [19]. Briefly; LTG molecule has 23 atoms and mixtures were classified into: (A) PVA physical mixtures
possesses C1 symmetry configuration. FTIR spectra of HPVA were analyzed for interaction by FTIR, which indicated an
and LPVA are consistent with literature [20]. Differences are overlapping of several peaks from various functional groups
reflected in total peak number, insignificant, (p≥0.05) shift of have led to insignificant shifting, (p≥ 0.05), in some peaks.
peak, and peak intensity. Two stretching bands appear in Overall, no interaction is detected since no new peaks
LPVA FTIR instead of one as in HPVA spectrum [21]. appeared, or other old peaks disappeared either. (B)
Similarities in molecular structure of Pluronic®F68 and Pluronic® physical mixtures had FTIR data that expressed no
Pluronic®F127, were reflected through FTIR absorption inter-composition interaction.
bands, where relatively broad peaks prevail due to their large

3.4. Formulation of LTG polymeric nanosuspension

Table 3: Polymeric LTG loaded nanosuspension preparations
Compositionh
Preparation
Surfactant
code 125 mg of biodegradable polymer type
Type Concentration (%w/v)
F1 0.5
PLCL (80PLA: 20PCL)
F2 4
HPVA
F3 0.5
PLCL (25PLA: 75PCL)
F4 4
F5 0.5
PLCL (80PLA: 20PCL)
F6 4
LPVA
F7 0.5
PLCL (25PLA: 75PCL)
F8 4
F9 1.25
PLCL (80PLA: 20PCL)
F10 2.5
Pluronic®F68
F11 1.25
PLCL (25PLA: 75PCL)
F12 2.5

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International Journal of Research in Pharmacy and Pharmaceutical Sciences

F13 1.25
PLCL (80PLA: 20PCL)
F14 Pluronic® 2.5
F15 F127 1.25
PLCL (25PLA: 75PCL)
F16 2.5
(h) Each formulation contained 25 mg LTG

3.5 Freeze-thaw test for selection of suitable F13), and finally (F16< F15). As per PS and PDI changes; PS
cryoprotectant significantly increased (p< 0.05) in all PVA-containing PNPs
Cryoprotectant effect of 5%w/v LPVA presented in table (4) as surfactant, while there was no significant PS change, (p≥
show that all systems were efficiently protected, with CF< 2. 0.05), in Pluronic®-containing PNPs as surfactant. This
There is significant difference (p< 0.05), in CF of (F2< F1), would indicate that Pluronics® might be improving the
(F4< F3), (F6< F5), (F8< F7), (F10< F9), (F12< F11), (F14< cryoprotectant effect of 5%w/v LPVA.

Table 4: Cryoprotectant activity of 5%w/v low molecular weight polyvinyl alcohol

Preparation PS before freeze-thawing PS after freeze-thawing PDI before freeze-thawing PDI after freeze-thawing
CF
symbol (Mean ± SD) (Mean ± SD) (Mean ± SD) (Mean ± SD)
F1 96.730 ± 11.610 152.300 ± 24.210 0.180 ± 0.002 0.276 ± 0.036 1.574
F2 261.800 ± 34.112 372.150 ± 39.870 0.274 ± 0.011 0.325 ± 0.023 1.422
F3 111.500 ± 17.461 165.500 ± 23.116 0.144 ± 0.014 0.177 ± 0.013 1.484
F4 310.700 ± 41.880 425.300 ± 49.002 0.146 ± 0.009 0.196 ± 0.021 1.240
F5 212.400 ± 42.100 321.100 ± 31.922 0.304 ± 0.025 0.362 ± 0.021 1.403
F6 265.800 ± 29.673 341.410 ± 29.542 0.213 ± 0.017 0.279 ± 0.030 1.284
F7 102.100 ± 13.562 152.400 ± 15.340 0.118 ± 0.009 0.160 ± 0.018 1.492
F8 140.000 ± 9.207 185.300 ± 13.452 0.145 ± 0.027 0.207 ± 0.013 1.323
F9 116.300 ± 20.012 204.400 ± 33.477 0.207 ± 0.013 0.236 ± 0.021 1.758
F10 94.550 ± 11.431 122.900 ± 14.111 0.185 ± 0.007 0.200 ± 0.016 1.406
F11 119.800 ± 21.094 155.300 ± 23.001 0.146 ± 0.004 0.172 ± 0.012 1.296
F12 74.920 ± 9.068 87.070 ± 10.720 0.158 ± 0.031 0.169 ± 0.003 1.162
F13 91.690 ± 18.001 112.100 ± 23.340 0.149 ± 0.010 0.162 ± 0.005 1.223
F14 78.680 ± 19.010 93.300 ± 15.972 0.124 ± 0.005 0.136 ± 0.012 1.186
F15 94.800 ± 8.973 111.100 ± 8.002 0.109 ± 0.017 0.121 ± 0.011 1.171
F16 92.870 ± 17.560 105.900 ± 13.681 0.108 ± 0.005 0.117 ± 0.020 1.140

3.6 Effect of type and concentration of cryoprotectant F4, F6, F8, F10, F12, F14, and F16. 5%w/v HPVA,
Optimization of both type and concentration of Pluronic®F127, and Pluronic®F68 were tested, with CF
cryoprotectant involves PNPs that showed significantly, (p< values presented and compared in table (5).
0.05), lower CF values, compared to their homologues: F2,

Table 5: Cryoprotectant activity of 5%w/v of several surfactants

CF values ± SD
Preparation symbol
HPVA Pluronic®F127 Pluronic®F68
F2 1.643 ± 0.011 1.188 ± 0.053 1.142 ± 0.021
F4 1.890 ± 0.009 1.182 ± 0.039 1.104 ± 0.019
F6 1.497 ± 0.017 1.213 ± 0.073 1.132 ± 0.029
F8 1.867 ± 0.027 1.285 ± 0.029 1.086 ± 0.017
F10 1.589 ± 0.007 1.133 ± 0.038 1.059± 0.022
F12 1.482 ± 0.031 1.108 ± 0.090 1.062± 0.009
F14 1.628 ± 0.005 1.119 ± 0.057 1.082± 0.011
F16 1.885 ± 0.005 1.081 ± 0.041 1.029± 0.019

All surfactants, resulted in CF<2, assuring their cryoprotectant for further investigations, as shown in table
cryoprotectant efficiency through CF values below “2”. (6). Results revealed that 10, 15 and 20%w/v Pluronic ®F68
Cryoprotectant effect of all surfactants was compared, have anti-aggregation effect, reflected in CF approximately
through their CF. Pluronic ®F68 is the most effective approaching value of “1”, and insignificant PS and PDI
cryoprotectant; showing minimum CF, while maximum CF changes. CF values at 5%w/v significantly (p< 0.05) varied
were resulted from HPVA, with significant difference (p< from 20%w/v, while no significant differences (p≥ 0.05)
0.05). Pluronic®F68 was thereby, used as model were noticed between CF at 15 and 20% w/v.

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Table 6: Cryoprotectant activity of increasing concentrations of Pluronic®F68

Cryoprotectant Factor (CF) on adding Pluronic®F68
Preparation symbol
5%w/v 10%w/v 15%w/v 20%w/v
F2 1.142 1.103 1.070 1.052
F4 1.104 1.062 1.043 1.028
F6 1.132 1.084 1.051 1.036
F8 1.086 1.048 1.034 1.021
F10 1.059 1.026 1.012 1.008
F12 1.062 1.038 1.020 1.013
F14 1.082 1.042 1.028 1.017
F16 1.029 1.011 1.002 1.002

3.7 Optimization of PNPs solvent systems, surfactant types and concentrations, and
Various parameters have been changed, by combining several polymers.
Table 7: Cloud point of ethanol in acetone- polymer solution
Polymer type CLalc (%) Acetone: ethanol ratio
PLCL (80PLA: 20PCL) 60 1: 1.5
PLCL (25PLA: 75PCL) 63 1: 1.7
Table 8: Cloud point between surfactant solution and drug-polymer solvent system
Surfactant concentration Solvent system: Surfactant solution CLbinary (%)
Surfactant type
(%w/v) Mono Binary Tertiary Mono Binary Tertiary
0.5 1: 1.5 1: 1.6 1: 1.7 60 62 63
1 1: 1.6 1: 1.6 1: 1.8 62 62 64
HPVA
2 1: 1.8 1: 1.7 1: 1.9 64 63 66
4 1: 2 1: 2 1: 2 67 67 67
0.5 1: 1.2 1: 1.3 1: 1.3 55 57 57
1 1: 1.4 1: 1.5 1: 1.4 58 60 58
LPVA
2 1: 1.7 1: 1.8 1: 1.16 63 64 62
4 1: 2 1: 2 1: 2 67 67 67
1 1: 1.5 1: 1.2 1: 1.6 60 55 62
Pluronic®F68
2 1: 1.8 1: 1.5 1: 1.9 64 60 66
1 1: 1.3 1: 1.2 1: 1.4 57 55 58
Pluronic®F127
2 1: 1.6 1: 1.4 1: 1.7 62 58 63

Alcohol percentage in polymer solution at cloud point, CLalc, indicated that solvent system has no effect on either
was recorded in table (7), as index of polymer affinity to morphology or PS, with all PNPs showing PS less than
solvent. As for surfactants; volume ratio of (solvent mixture: 200nm, which is optimal for passing BBB without getting
surfactant solution) at cloud point, CLbinary, was determined in caught by the RES. Figure (2) shows the effect of varying
table (8), as index of surfactant affinity to solvent mixture. surfactant concentration and molecular weight on
3.8 Transmission Electron Microscopy (TEM) morphology and PS through investigating
In figure (1), effect of varying solvent system is studied [LTG/HPVA/PLCL (25: 75)] and [LTG/LPVA/PLCL (25:
through [LTG/HPVA/PLCL (80: 20)] PNPs; where results 75)] formulations. No considerable change was noticed.

MP1H0.5 BP1H0.5 TP1H0.5 MP1H1 BP1H1 TP1H1

MP1H2 BP1H2 TP1H2 MP1H4 BP1H4 TP1H4

Fig 1: TEM of [LTG/HPVA/PLCL (80: 20)] polymeric nanoparticles
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International Journal of Research in Pharmacy and Pharmaceutical Sciences

MP2H0.5 MP2H1 MP2H2 MP2H4

MP2L0.5 MP2L1 MP2L2 MP2L4

Fig 2: TEM of [LTG/HPVA/PLCL (25: 75)] and [LTG/LPVA/PLCL (25: 75)]

MP1F1 MP1F2 MP1F3 MP1F4 MP1F5 MP1F6

MP1F7 MP1F8 MP1F9 MP1F10

Fig 3: TEM of [LTG/Pluronic®/PLCL (80: 20)] polymeric nanoparticles

BP2F1 BP2F2 BP2F3 BP2F4 BP2F5

BP2F6 BP2F7 BP2F8 BP2F9 BP2F10

Fig 4: TEM of [LTG/Pluronic®/PLCL (25: 75)] polymeric nanoparticles

Figures (3) and (4), showing TEM of Pluronic® based PNPs, changing polymer or surfactant type or even (polymer:
illustrating effect of changing polymer and surfactant type, as surfactant) ratios, since all particles appeared to be spherical
well as (polymer: surfactant) ratio. Results reveal no effect of in shape with size lying below 200nm.

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International Journal of Research in Pharmacy and Pharmaceutical Sciences

4. Discussion cryoprotectant that would be used [29]. Results preliminarily

4.1 Assessment of calibration curves for lamotrigine suggest that: as total content of surfactant increases, the
All R2 values calculated are compatible with the “ideal R2 whole system becomes more protected against aggregation.
model” interpretation. Also, values of |r| were all > 0.7, Unlike conventional cryoprotectants that inhibit freezing by
indicating a strong correlation between the two variables17; interacting with water, ice blockers as PVA, act by direct
drug concentration and absorbance (as reflection of molecular recognition of ice nucleators [30]. This selective
solubility). R2 values generally ranged between 0.992 and attraction to ice growth surfaces permits PVA to exert
0.999 (± SD). These results reflect a strong linear component significant effects while present at very low molecular
of the presented models. weights and concentrations [31], thus, protect systems against
aggregation without additional toxicity.
4.2 Determination of thermodynamic solubility
Acetone would be solvent of choice for mono-solvent system,
as it solubilizes both drug and polymers. But, single solvent 4.5 Effect of type and concentration of cryoprotectant
would not promise optimum solubilization, so, cosolvent was Pluronic®F68 is the most effective cryoprotectant; showing
suggested. Since, cosolvency power is stronger for water- minimum CF. Pluronics® are reputed as one of the most
miscible alcohols, because; as explained by Ladaa et al. as effective stabilizers. Steric stabilizers, as Pluronics® act by
carbon chain length of alcohols increases, their cosolvency steric hindrance and shielding particles’ surface against
power increases, thereafter; ethanol would be preferred to freezing stresses, thus decreasing hydrogen bonding between
methanol, as it enhances solubility of hydrophobic drugs as particles [32], preventing their agglomeration. Concentration-
LTG, as most of cosolvents contain hydrogen bond donor dependent cryoprotection was observed through increase in
and/or acceptor groups with little part of hydrocarbon PS at lower cryoprotectant concentrations. Increase in PDI
regions, in addition to interfering with water’s self- was also noticed, due to cryoprotectant micelles formation40.
association and decreasing water’s ability to pull out non-
polar hydrophobic components [24]. In addition, DCM shows 5. Conclusion
maximum solubility to polymers, and minimum solubility to Various preformulation studies were carried out starting by
LTG, thereby; it might be used as secondary cosolvent in UV absorbance studies, whose data were a guide to LTG
combination with alcohol. solubility in various solvents. Acetone was chosen as the
mono solvent to solubilize both LTG and polymers, while
4.3 Compatibility study using Fourier transform infrared ethanol was the cosolvent of choice, and finally DCM was
(FTIR) the subsidiary cosolvent in a tri-solvent system. Pluronic®F68
IR bands of PVA are quite broad and overlapped in “600 to was the most suitable cryoprotectant capable of producing
1500” cm-1 region, due to: (1) half PVA bulk consists of dried re-suspendable stable preparations, with no significant
crystallites embedded in glassy matrix, where difference of increase in either particles size or PDI. Finally, TEM was an
symmetry between free and unfree PVA molecules evidence of the success of preformulation studies in
contributes to two sets of frequencies [25]. (2) slight shift achieving spherical LTG-loaded stable PNPs formulations
between equivalent frequencies in these two phases whose particle size is less than 200nm to assure their
contributes to overlapping. (3) PVA possesses an intended parenteral administration for sustained central anti-
“intermediate” region other than crystalline and amorphous epileptic effect.
phases. Thus; these regions show a mixed symmetry due to
random placement of OH groups along the chain, resulting in 6. References
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3. Castel-Branco MM, Almeida AM, Falcao AC, Macedo
4.4 Freeze-thaw test for selection of suitable TA, Caramona MM, Lopez FG. Lamotrigine analysis in
cryoprotectant blood and brain by high-performance liquid
Freeze-dried suspensions usually show strong tendency to chromatography. J Chromatogr B Biomed Sci
agglomerate after drying, due to phase separation during Appl. 2001; 755(1-2):119-127.
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