Improved Stability and Enhanced Oral Bioavailability of Atorvastatin Loaded Stearic Acid Modified Gelatin Nanoparticles

Deepanshu Shilpi#1, Varun Kushwah#1, Ashish Kumar Agrawal1,2, Sanyog Jain1*

# Authors contributed equally

1Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics,National Institute of Pharmaceutical Education and Research,

SAS Nagar, Punjab – 160062, India.

2James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA

* To whom correspondence should be addressed: E-mail: sanyogjain@niper.ac.in,sanyogjain@rediffmail.com, Tel.: +91-172-2292055, Fax: +91-172-2214692

Conflict of Interest and DisclosureThe authors report no financial interest that might pose a potential, perceived, or real conflict of interest.

All sources of support for research:The authors are thankful to the Director, NIPER for providing the necessary infrastructure and facilities. Varun Kushwah and Ashish Kumar Agrawal are grateful to Council of Scientific and Industrial Research (CSIR), GOI, New Delhi, for providing research fellowships.

1 Methods1.1 In-vitro HPLC method development and validation

The samples were analysed by using validated HPLC method for the quantification of AT.

Isocratic and gradient method at a flow rate of 1ml/min was developed for the In-vitro and

In-vivo analysis of the AT, respectively. Further the detailed parameters of validated HPLC

method are mentioned below:

Table 1: In-vitro HPLC method validation parameter

Parameters System Condition Column Waters spherisorb 5µm C8 Elution Isocratic elution Mobile Phase Acetonitrile: phosphoric acid buffer (pH 2.9)Run time 12 min Flow rate 1 mL/min Detection wavelength 244 nm Temperature 30ºC Sample injection volume 20 µL

Table 2: In-vitro HPLC method validation parameters

Parameters Values Linearity Range 5-25 µg/mL Correlation coefficient 0.998±0.0005 Slope 98288±1026 Intercept 40908±20489 LOD 0.269 µg/mL LOQ 0.817 µg/mL Values are in mean ±SD (n=3)

Table 3: Estimated intra-day and inter-day precision and accuracy of the method

Concentration(g/mL)

Intraday accuracy (%)

Interday accuracy (%)

Intraday precision

Interday precision

16 99.68±0.07 99.80±0.27 1.43 1.5020 99.77±0.31 99.74±0.44 1.02 1.2224 96.46±4.90 99.12±0.16 1.59 1.66

Values are in mean±SD (n=3)

1.2 In-vivo HPLC method development and validation

Critical factors such as ratio of mobile phase compositions, pH and flow rate affecting the

elution and peak pattern were optimised. A calibration curve was plotted in the range of 100

to 1000 ng with linearity and R2 value of 0.988 using the gradient elution mode.

Chromatograms of Atorvastatin and IS (internal standard) were obtained by simultaneous

elution in gradient mode at 244 nm respectively. Atorvastatin was eluted at RT of 7.30 min.

and IS at 9 min. The details are given in the table below.

Table 4: In-vivo HPLC method chromatographic conditions

Parameters System conditionsColumn Waters spherisorb 5µm C8Elution Gradient elution Mobile Phase A=Acetonitrile, B= Phosphoric acid buffer (pH 2.9)

Time A% B%0 50 508 90 1015 50 5020 50 50

Flow Rate 1 mL/minDetection Wavelength 244nmTemperature 30°CSample Volume 30 µlRetention time 7.30min

Values are in mean ±SD (n=3)

Table 5: In-vivo HPLC method validation parameter

Parameters ValuesLinearity Range 0.1-1 µg/mLCorrelation coefficient 0.988±0.0015Slope 0.605±0.00661Intercept 0.0233±0.0037LOD 0.033 µg/mLLOQ 0.1 µg/mL

Values are in mean ±SD (n=3)

Table 6: Estimated intra-day and inter-day precision and accuracy of the method

Concentration (g/mL)

Intraday accuracy (%)

Interday accuracy (%)

Intraday precision

Interday precision

0.1 97.50±0.02 97.70±0.72 0.61 0.570.5 99.72±0.28 99.69±0.19 0.41 0.691 97.59±0.39 97.59±0.44 0.52 0.43

Values are in mean ± SD (n=3)

1.3 Spectral Characterization of Stearic acid conjugated gelatin (MG)

The synthesis of SA-gelatin conjugate is detailed in two steps comprising: (i) preparation of

active NHS ester of SA using standard carbodiimide chemistry; (ii) covalent conjugation of

activated ester of SA with gelatin. Synthesis of SA-gelatin conjugate (modified gelatin) was

confirmed by proton NMR (1H NMR) analysis. Briefly, synthesized conjugate was dissolved

in D2O and 0.1% TMS used as an internal reference. The 1H NMR evaluation of developed

conjugate was performed using a Bruker BioSpin (Fallanden, Switzerland) 400 MHz

spectrometer and the spectral plotting was acquired using Bruker’s TOPSPIN 2.1.

Furthermore, Fourier transform infrared spectroscopy (FT-IR) analysis of SA-gelatin

conjugate was also analysed using a Perkin Elmer, FT-IR spectrometer, USA. Briefly, the

sample were pressed into KBr pellet and the spectra were recorded over a range of 4000-450

cm-1.

1.4 Optimisation of AT loaded hybrid nanoparticles

Different process variables such as effect of different solvents, solvent volume, surfactants,

cross linking agent, stirring speed (600, 1200, 1800), and % theoretical drug loading (5-20%)

were extensively optimized, in order to obtain the final formulation with optimum

formulation parameters in terms of particle size, Polydispersity Index (PDI) and Entrapment

efficiency (Table 7).

Table 7: Optimization parameters and different process variables for the preparation of AT

loaded MG NPs.

S. No. Optimization parameters Variables Remarks

1 Solvent type Methanol Acetone Ethanol

Solvent Volume: 6Stirring speed: 1200 rpmSurfactant: Lutrol F-108Surfactant concentration: 0.5% w/vCrosslinker:PAA (1% v/v)

2 Solvent volume of acetone (ml)

2 3 4 5 6 7 8

Solvent: AcetoneStirring speed: 1200 rpmSurfactant: Lutrol F-108Surfactant concentration: 0.5% w/vCrosslinker:PAA (1% v/v)

3 Stirring speed (rpm) 600 1200 1800 2000

Solvent: AcetoneSolvent Volume: 6Surfactant: Lutrol F-108Surfactant concentration: 0.5% w/vCrosslinker:PAA (1% v/v)

4 Surfactant type Lutrol F-127 Lutrol F-108 Tween-80

Solvent: Acetone Solvent Volume: 6Stirring speed: 1200 rpmSurfactant concentration: 0.5% w/vCrosslinker:PAA (1% v/v)

5 Surfactant conc.(mg) 0.5% w/v 1.0% w/v 1.5% w/v2.0% w/v

Solvent: AcetoneSolvent Volume: 6Stirring speed: 1200 rpmSurfactant: Lutrol F-108Crosslinker:PAA (1% v/v)

6 Cross linking agent type PAAPSS

Solvent: AcetoneSolvent Volume: 6Stirring speed: 1200 rpmSurfactant: Lutrol F-108Surfactant concentration: 0.5% w/v

1.5 Degree of conjugation

The extent of cross-linking of SA with gelatin was monitored by assessing the surface free

amino groups using 2,4,6 trinitrobenzenesulfonic acid (TNBS) method. Briefly, 0.01%

TNBS solution was added to the 0.25 mg/mL samples (gelatin and MG) in 0.1 M sodium

bicarbonate buffer (pH 8.5). The reaction mixture was kept in dark for 2 h at 40°C. Finally, 2

mL SDS 10% and 1 mL HCl (1 N) were added to each sample, mixed, and the absorbance of

each solution was measured at 340 nm using UV/Vis spectrophotometers (Beckman Coulter,

DU®730). The standard calibration curve was made by a series of known L-lysine

concentrations using the TNBS method. Further the calibration curve was utilized to

calculate the molar concentration of free amino groups in modified gelatin (MG) and native

gelatin (NG) and the degree of conjugation (DC) was calculated as follows:

DC=C NG−CMG/CNG×100

Where, CNG and CMG are the concentration of free primary amino of NG and MG,

respectively

2 Results2.1 Spectral Characterization of Stearic acid conjugated gelatin (MG)

2.1.1 1H NMR spectroscopy

The covalent bond between stearic acid and gelatin was confirmed by 1H NMR spectroscopy.

In the 1H NMR study, processed gelatin was also used to compare with MG. The 1H NMR

spectrum of MG Post modification showed additional chemical signal was observed at 1.2

ppm (CγH2) and at 2.2 ppm (COCαH2) indicating the protons of Stearic acid group

(Chemical linkage of the hydrophobic stearic acid group on the gelatin backbone). A

chemical shift at 3.2 ppm (CONH) was observed indicating a successful replacement of the

hydrophobic group by the amino group along the gelatin molecules. Which suggested the

amide reaction between the carboxyl group of stearic acid and the gelatin amino groups.

(A)

(B)

Figure 1: 1H NMR of (A) native gelatin (B) modified gelatin

2.1.2 FT-IR Spectroscopy

FT-IR is a widely used technique to detect the structural changes as well as confirm the

complex formation and chemical interaction between functional groups. In the reaction of

gelatin and stearic acid, the most characteristic bands in FT-IR spectra are associated with the

amide groups including typical C=O stretching at 1630–1695 cm -1, for amide I, N-H

associated with the C=O stretching vibration at 1600– 1700 cm-1 for the amide II, and N-H

stretching at 3000–3500 cm-1 for amide A. The FT-IR spectra showed significant differences

between MG and processed gelatin. For instance, MG had a peak that stands for N-H stretch

at 3409 cm-1, whereas the gelatin peak was at 3357 cm-1. In addition, the gelatin N-H stretch

peak was broader than that of MG because gelatin amino groups (2 N-H stretches) were

reacted to form an amide group (1 N-H stretch). While there was appearance of a new N-H

bend at 1536 cm-1 in the MG spectrum may prove the presence of new amide groups made by

the amide reaction between stearic acid and the amino groups of gelatin.

MG

NG

Figure 2: FT-IR of modified gelatin (MG) and native gelatin (NG)

2.2 Optimisation of AT loaded hybrid nanoparticlesThe critical parameters for the development of MG nanoparticles were carefully optimized

and their influence on particle size, PDI, and entrapment efficiency was analysed. The

concentration of gelatin was kept constant i.e. 150 mg throughout the experiment. Acetone

was found as the ideal solvent with volume of 2-8 ml (6 ml) providing a particle size of

342.40±11.661nm and PDI of 0.340±0.096. Furthermore, the stirring speed at 1200 rpm

yielded NPs with optimum parameters of size and PDI. At lower stirring speed (600 rpm),

formulations of higher particle size (266.26±25.64 nm) and PDI (0.571±0.45) were obtained,

while at higher stirring speed at 1800 and 2000 rpm, PDI of the NPs were found to be in

optimum range (<0.3), but particle size was found to be higher as compared to size obtained

at 1200 rpm. The effect of various surfactant and their concentration on particle size and PDI

of MG-NPs were also evaluated, which showed optimum parameters of NPs with Lutrol F-

108 with a concentration of 0.5% w/v. Furthermore, NPs developed via utilizing PAA (cross

linker) at a 0.1% v/v exhibited smaller particle size and PDI, compared to PSS.

After extensive optimization of AT loaded NPs, the % EE of AT in NPs were also evaluated.

As a result of enhanced hydrophobicity of MG, by the virtue of SA conjugation, MG-NPs

exhibited enhanced %EE compared to NG NPs. Although, MG NPs showed highest

entrapment efficiency of 86.40±4.85 at 5% theoretical loading, the practical loading was

found to be very less, i.e., 4.32±0.24 practical loading. While, the highest practical loading

was achieved in case of 20% theoretical drug loading with particle size of 0.249±0.07 nm

and %E.E of 58.70±5.37%.

Table 8: Optimization of parameters influencing particle size and PDI

S. No. Optimization parameters Variables Particle size (nm)

PDI

1 Solvent type Methanol Acetone Ethanol

2256.5±755.50 342.40±11.661 1278.5±363.58

0.490±0.65 0.340±0.09 0.438±0.12

2 Solvent volume of acetone (ml) 2 3 4 5 6 7 8

40.43±15.08 162.40±11.66 189.96±11.57 210.06±18.02 237.23±36.49 267.43±26.60370.57±28.34

0.066±0.06 0.104±0.09 0.187±0.070.374±0.35 0.209±0.10 0.440±0.05 0.436±0.02

3 Stirring speed (rpm) 600 1200 1800 2000

266.26±25.64 179.23±16.39 242.83±19.25 230.42±18.24

0.571±0.45 0.186±0.02 0.166±0.02 0.198±0.02

4 Surfactant type Lutrol F-127 Lutrol F-108Tween-80

1379.66±243.85 196.23±28.53 83.27±19.01

0.551±0.07 0.203±0.09 0.434±0.02

5 Surfactant conc.(mg) 0.5% w/v 1.0% w/v 1.5% w/v2.0% w/v

186.27±23.51 192.89±26.49 186.45±16.52 206.37±31.78

0.193±0.10 0.199±0.01 0.196±0.03 0.205±0.05

6 Cross linking agent type PAA PSS

166.23±46.49 230.28±36.52

0.185±0.01 0.256±0.03

Table 2: Optimization of Entrapment Efficiency (% E.E)

Theoretical Drug loading (%)

Particle Size PDI (nm)

% EE Practical loading

AT loaded MG NPs5 178.23±18.69 0.197±0.03 86.40±4.85 4.32±0.24 10 181.97±31.00 0.193±0.08 70.31±3.85 7.03±0.38 15 205.83±7.32 0.161±0.09 61.32±4.80 9.19±0.72 20 247.73±10.95 0.219±0.07 58.70±5.37 11.74±1.07 25 378.33±12.79 0.437±0.05 30.21±3.33 8.4±0.87 AT loaded NG NPs 5 189±11.03 0.144±0.103 28.8±21.8 1.44±1.09 10 202±14.22 0.175±0.04 18.25±4.65 1.84±0.58 2.3 Degree of conjugation

The degree of conjugation was estimated by evaluating free –NH2 groups in NG and MG by

the TNBS method. The content of amino group in the L-lysine in the concentration range of

1 to 20 μg/mL was calculated and used as a reference. The molar concentration of free amino

groups in NG and MG were 158.00 ±5.65 μmol and 46.65 ± 6.85 μmol, respectively (Table

9). The corresponding reduction of the amino groups in MG compared to NG was a result of

conjugation of SA via amide bond between carboxylic group of SA and amine group of NG.

Assuming amino groups of NG as 100%, the degree of conjugation of SA was found to be

70.47%.

Table 9: Degree of conjugation determined via 2,4,6-trinitrobenzene sulfonic acid (TNBS) method

NG (μmol) MG (μmol)Free amino group concentration 158.00 ±5.65 46.65 ± 6.85Degree of conjugation (DC) % 70.47%

Values represent mean ± SD (n = 6)