Formulation and Characterization of Tenofovir Disproxyl Fumerate Nanoparticles prepared by Nanoprecipitation Method

 

S. Sivaprasad1*, Ch. Sadakvali2, V. Ravi Kumar1, P.V. Murali Krishna1,

Shaik Mohammed Yusuf 3, A. Srikanth4

1MNR College of Pharmacy, MNR Higher Education and Research Academy Campus,

Fasalwadi (V), Sangareddy, Telangana, India, 502294.

2Mohammadiya Institute of Pharmacy, Khammam, Telangana.

3Department of Pharmacy, College of Health Sciences, Debre Tabor University, Ethiopia.

4Lovely Professional University, Punjab.

*Corresponding Author E-mail: sagiliprasad2003@gmail.com

 

ABSTRACT:

Tenofovir disproxyl fumerate (TDF) is widely used drug in anti HIV treatment. The main disadvantage with TDF is its low bioavailability. To overcome this problem TDF is formulated in to nanoparticles by using nanoprecipitation method. The formulated nanoparticles were in the range of 106.8nm to 516.4nm. The effect of PLGA and TPGS on the formulated nanoparticles were also studied. It is evident that the concentration of PLGA played an important role in the entrapment and drug loading capacity. On the basis of drug loaded and entrapment efficiency the F8 formulation was considered as optimized formulation. Different analytical techniques were used to determine the amount of drug present in the optimized formulation. The optimized formulation (F8) was subjected to different drug release kinetic models and its stability studies were also conducted in accordance with ICH guidelines.

 

KEYWORDS: Tenofovir, Anti HIV treatment, PLGA, TPGS, Nanoprecipitation method.

 

 


INTRODUCTION:

Tenofovir disproxyl fumerate (TDF) is one of the widely used first line drug in the treatment of human immune deficiency virus (HIV). It is a potential inhibitor of virus nucleotide reverse transcriptase enzyme. It is less toxic to the body and longer half life i.e. 17 hours made this drug suitable for long term treatment in HIV patients. Apart from the advantages mentioned above it suffers from the draw backs of lesser oral bioavailability. Therefore formulating the drug in to nanoparticles is obvious for improving the bioavailability of tenofovir1. Nanoparticles posses several advantages over conventional dosage form. There is minimal drug wastage during transit through the GI tract and escapes degradation in the acidic environment of stomach.

 

The typical size and other surface characters of nanoparticles allows easy uptake by the M cells in the peyer’s patches without interrupting the integrity of nanoparticles2. The lymph tissue presented in the patches facilitates distribution of the nanoparticles through the systemic formulation and hence extends the half life of the drug and sustained drug release can be achieved. The other benefits with nanoparticles are reduction of therapeutic dose, increased bioavailability and reduced toxic effects. Selection of polymer is a crucial step in the manufacturing of nanoparticles. Among the biodegradable polymers available in the market. TPGS (d-alpha-tocopheryl polyethylene glycol) is widely used novel material to achieve high drug encapsulation efficiency and desirable Pharmaco kinetic properties of the drug loaded nanoparticles. The aim of the present study is formulation of nanoparticles by nanoprecipitation method and to determine the effect of surfactants and bio degradable polymers in assessing different parameters like entrapment efficiency, drug loading ratio and in vitro studies on the formulated tenofovir nanoparticles.

MATERIALS AND METHODS:

The TDF drug was obtained as a gift sample from Matrix pharmaceuticals limited, Hyderabad. Acetone (HPLC grade) was supplied by Merck India; Water by Milli-Q, Millipore, Mumbai, TPGS was obtained as a gift sample form Eastman Company, UK. PLGA (50:50) polymer was purchased from Boehringer Ingelheim, Germany. Polyvinyl alcohol (30000 to 70000) molecular weight was purchased from Sigma- Aldrich Chemicals Private Limited, Bangalore.

 

Preparation of nanoparticles2:

TDF Nanoparticles containing PLGA as a polymer were prepared by nanoprecipitation method. In this method 5mg of the TDF and varying quantities of PLGA (50:50) were dissolved in 3 ml of acetone (Solution A). Different concentrations of TPGS were dissolved in 10 ml of deionized water (Solution B). Solution A was added to Solution B using a syringe at the flow rate of 1 mL/10 min by magnetic stirring (1500rpm) at room temperature. From above method, eight different batches of TDF nanoparticles were prepared with various concentrations of PLGA and coded as F1–F8. The obtained nanosuspension was centrifuged, lyophilized and subjected for various characterization parameters (Sangeeta Mohanty et al., 2019)

The first part of the plan of work was to optimize the concentration of surfactant to be used in the formulation of nanoparticles. To achieve this, the first three formulations were planned with TPGS concentrations 0.015%, 0.03% and 0.06% respectively. The optimization of surfactant concentration was done on the basis of particle size and entrapment efficiency of nanoparticles obtained.

 

As the least particle size and best entrapment efficiency was obtained for F2 formulation when compared to F1 and F3, it was decided that the 0.03% of TPGS was the optimum concentration to be used in further formulations. The next part of the plan of work was to optimize the drug polymer ratio. For this, 5 batches were planned (F4 to F8) using the drug polymer ratios of 1:5, 1:10, 1:15, 1:20 and 1:25 respectively. The optimum drug polymer ratio was selected on the basis of entrapment efficiency of the polymer. (Adnan M. Jasim. et al., 2019)

 

The composition of the TDF nanoparticles and drug polymers ratios were given in Table1 and 2 respectively.

 


 

Table 1 Composition of Tenofovir nanoparticles

Ingredients

Batch no

F1

F2

F3

F4

F5

F6

F7

F8

PLGA (50:50)(mg)

13

13

13

25

50

75

100

125

TPGS(%g/ml)

0.015

0.03

0.06

0.03

0.03

0.03

0.03

0.03

Tenofovir (mg)

5

5

5

5

5

5

5

5

Acetone (ml)

3

3

3

3

3

3

3

3

Water (ml)

10

10

10

10

10

10

10

10

 


Table 2 Formulations used for Entrapment efficiency, drug loading capacity and in vitro studies

Ingredients (mg)

F6

F7

F8

PLGA (50:50)(mg)

75

100

125

TPGS(%g/ml)

0.03

0.03

0.03

Tenofovir (mg)

5

5

5

Acetone (ml)

3

3

3

Water (ml)

10

10

10

 

METHODS:

Drug polymer compatibility studies3

Compatibility of pure drug with excipients was determined by carrying out FTIR studies. Infrared spectrums of TDF, PLGA, TPGS and physical mixture of drug and polymer was determined on Perkin Elmer FTIR Spectrophotometer, series 1600 which was calibrated with polystyrene using KBr dispersion method in the region between 400‐4000 cm‐1.

 

Differential scanning calorimetry4 (DSC):

The thermal analysis was performed using a Mettler Toledo star system 822e differential scanning calorimeter, equipped with liquid nitrogen cooling system. The machine was calibrated with pure indium (M.P.1550C) and zinc (M.P.419.50C) as standard calibrators prior to experiments. Samples, API and drug excipient mixed powders (5-8mg) were crimped in aluminium pans with lids and tested under dry evaporation of any water or residual solvent existing in the sample. The test was conducted with scanning rate of 10oC/min over the temperature range of 50oC-250oC for drugs/excipients/mixtures. An empty aluminium pan was used as a reference.

 

Physico Chemical Evaluation:

Particle size analysis and zeta potential measurement5

Particle size of the nano formulation was very important because the parameters like biological fate, distribution in the body fluids and other physico chemical properties were depends on particle size. The nanoparticles size was determined by dynamic light scattering technique (DLS) using Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK). This technique measures the time dependent fluctuations in the intensity of scattered light which occurs due to particles in constant Brownian motion. (Jessy Shaji. et al., 2018)

 

Zeta potential represents the stability of the nanoparticles dispersion. Each sample was diluted with phosphate buffer (pH 7.4) and the surface charge (zeta potential) of the NPs determined by measuring their electrophoretic mobility of the NPs by the zeta sizer (Malvern Instruments, UK).

 

Surface morphology6

The Tenofovir loaded nanoparticles were subjected to Scanning Electron Microscope (SEM) analysis for assessing its size and shape. (S Rajarajan. et al., 2009)

Drug Entrapment Efficiency

 

Lyophilized nanoparticles 3mg were dissolved in 1ml of diluents and the drug amount was determined by HPLC analysis. The encapsulation efficiency was determined as the mass ratio of entrapped tenofovir in nanoparticles to the theoretical amount of the drug loaded in the nanoparticles. The entrapment of the Tenofovir PLGA nanoparticles was expressed as loading capacity. (Jin-Wook Yoo. et al., 2011).

 

                          Amount of the drug in the nanoparticles

Entrapment  = ------------------------------------------------------------ × 100

Efficiency (%)      Amount of the drug loaded in nanoparticles

 

In vitro release studies7,8

The in vitro studies were carried out by using dialysis bag technique. 10mg drug equivalent freeze dried TDF loaded nanoparticles were dispersed in 3 ml pH 7.4 phosphate buffer solution which is transferred in dialysis bag and suspended in 100 ml of isotonic pH 7.4 Phosphate buffer solution (PBS). The bag was placed under magnetic stirring in a water bath maintained at 37 ± 0.5° C. At fixed time intervals 5ml of samples were taken out and fresh buffer was replaced. The obtained solution was analyzed by HPLC to determine the drug content. (Vinod Kumari. et al., 2019)

 

Kinetic modelling9

In order to understand the kinetics and mechanism of drug release, the result of in vitro drug release study of nanoparticles were fitted with various kinetic equations like zero order (cumulative % release vs. time), first order (log % drug remaining vs. time), Higuchi’s model (cumulative % drug release vs. square root of time), Peppas model (log % drug release vs. log time). Coefficient of determination (R2), rate constant (K), diffusion coefficient (n) values were calculated for the linear curve obtained by regression analysis of the above plots. (D. Maheswara Reddy. et al., 2021).

 

Stability studies10

Stability of the prepared TDF nanoparticles was determined as per the International Conference on Hormonization (ICH) QIA (R2) guidelines (S. Sivaprasad. et al., 2020) to assess its physical appearance, drug content and release kinetics. Tenofovir loaded nanoparticles were packed separately in screw capped HDPE bottles sealed with aluminium seals which are stored at 45OC/75% relative humidity (RH) in the stability chamber (REMI, Mumbai, India) for 6 months. The samples were collected at 0, 1, 3 and 6 months. During this period the samples were tested for physical appearance, drug content and drug release studies. (Shyam S Kumar. et al., 2021) (Urvashi Jain. et al., 2021)

 

Statistical analysis:

All experiments were repeated at least 3 times. Data are expressed as a mean ± standard deviation (SD, n = 3)

 

RESULTS AND DISCUSSION:

FTIR spectra were used to identify the functional groups in TDF, PLGA (50:50) and TDF-PLGA 50:50 formulations. This is to determine the susceptibility these functional groups to various chemical reactions. Fig. 1, 2 and 3 represents the spectra of TDF, PLGA 50:50 and combination of TDF-PLGA 50:50. The FTIR spectra of these mixtures were compared with Pure TDF and PLGA 50:50 to identify the differences like appearance and disappearance of such functional groups in the combination. TDF (Fig.4) showed a characteristic peak at 34.59.12 cm-1 denotes the N-H stretching vibration bands of amino group. Intensity peaks at 2981.38 cm-1 and 2814.22 cm-1 were due to hydroxyl (OH) stretching. The band at 2084.44 cm-1 due to C=C, and the intensity peaks at 1752.31 cm-1 and 1671.87 cm-1 were due to C=O. The pure PLGA 50:50 (Fig.5) showed a characteristic peak at 2950.52 cm-1 due to the -OH stretching vibrations. The peak at 1750.32 cm-1 due to the C=O stretching of the carbonyl group. The TDF-PLGA 50:50 physical mixture (Fig.6) showed a characteristic peak at 2993.81 cm-1, due to O-H bridge stretching and a peak at 1750.07 cm-1 due to C=O stretching.

 

The FTIR spectra of TDF-PLGA 50:50 physical mixtures showed a slight variations in band lengths when in comparison to individual components which indicates the there might be a physical interaction between the TDF and PLGA 50:50.

 

 

Fig: 1. FTIR spectra of pure TDF

 

Fig: 2. FTIR spectra of PLGA 50:50

 

 

Fig: 3. FTIR spectra of TDF-PLGA 50:50

 

DSC spectroscopy:

In the thermogram of TDF two endothermic peaks were observed, within the range of temperatures, 111°C-118°C; these peaks represents the melting points of two different polymorphic forms of TDF, from the literature it is evident that there are about three different polymorphic forms (A, B and I). The first endothermic peak was because of polymorphic form I melting, which was then followed by recrystallisation (an exothermic event) of the melted API into form I which presented with a melting peak of 118°C, thus both endothermic peaks can be attributed to the different forms of TDF.

 

 

Fig: 4. DSC spectra of Tenofovir disproxyl fumerate

 

 

Fig: 5. DSC spectra of PLGA

 

 

Fig: 6. DSC spectra of TDF-PLGA 50:50

 

Fig. 6 represents the DSC spectra of TDF when it was analyzed as a physical mixture by mixing with PLGA 50:50. A single endothermic peak at 58.27oC was observed. In comparision to PLGA alone, a slight shift of about 3-4 oC was observed in the mixture. This indicates increased stability of physical mixture.

 

HPLC method:

The samples which are collected durindg diffusion studies were analyzed by HPLC technique. For this purpose standard plot was plotted by using the reference standard of TDF.

 

Fig: 7. Standard graph for TDF nanoparticles

 

Evaluation of TDF nanoparticles

Measurement of Particle size, Zetapotential, Drug loading and Entrapment Efficiency (EE).

The prepared formulations were evaluated for its particle size distribution, zeta potential and entrapment efficiency. The particle size of all the formulations were ranging from 106.8 nm to 516.4 nm. The drug entrapment efficiency of different formulations were in the range of 4 % to 98.8%. Zeta potential which is a measure of stability in the range of -0.276 mV to -27.2 mV. All the values were tabulated below (Table.3)

 

From the values it is evident that there was a gradual increase in the entrapment efficiency and decrease in the particle size on increasing the polymer concentration in the formulation composition. Based on the EE values the formulations F6, F7 and F8 were taken in to consideration for in vitro release studies.

 

The SEM images of TDF formulation indicates that the nanoparticles were spherical and no agglomeration were seen.

 

Table: 3 Evaluation Studies of Prepared Nanoparticles: Entrapment Efficiency, Particle size, Zeta Potential and Drug Loading

Batch No

Particle size (nm)

Zeta potential (mV)

Drug

Loaded (mg)

Entrapment

Efficiency (%)

F1

249.8

-0.276

0.20

4.0

F2

150.5

-5.12

0.24

4.8

F3

516.4

-1.98

0.22

4.4

F4

120.5

-4.17

1.24

24.8

F5

106.8

-24.1

1.86

37.2

F6

132.3

-24.7

3.08

61.6

F7

155.5

-25.6

4.16

83.2

F8

122.4

-27.2

4.94

98.8

 

 

Fig: 8. SEM image of TDF optimized formulation

 

In vitro release studies:

The selected formulations F6, F7 and F8 which were having drug: polymer ratios 1:15, 1:20 and 1:25 shown the drug release after 240 hrs in pH 7.4 phosphate buffer was found to be 96.18%, 92.14% and 86.14% respectively. These results were shown that the nanoparticles were able to sustain the release of TDF by showing slow absorption rate and therefore suitable for controlled release of drug. Hence the F8 formulation was considered as optimized formulation. The drug release profiles of F6, F7 and F8 were shown in Fig. 9. The mechanism of TDF release from the nanoparticles was evaluated by fitting the release data to 4 mathematical models namely; zero order, first order, higuchi and korsmeyer-peppas model. All the kinetic models were shown in fig. no.10 and the results were shown in table no. 4. On the basis of R2 value it is concluded that the optimized formulation F8 follows higuchi release kinetics. The data suggest that drug entrapped outside the nanoparticles released immediately followed by polymer breaking down and releasing the entrapped drug.

 

Table: 4 Diffusion study profiles for F6, F7, F8

Time (Hr)

Cumulative % drug release

F6

F7

F8

0

0

0

0

24

34.24

32.46

25.63

48

41.21

39.43

32.19

72

47.98

44.72

39.62

96

56.12

52.24

46.62

120

63.34

56.42

49.55

144

66.89

64.76

55.57

168

75.82

69.86

64.24

192

80.67

75.45

68.24

216

87.26

81.23

75.45

240

96.18

92.14

86.14

 

 

Fig: 9. Diffusion study profile Cumulative % release Vs Time (hrs)

 

 

Fig: 10. Drug release kinetic plots for optimized formulation-F8

Table: 5 Interpretation of R2 values and the rate constants of release kinetics of nanoparticles.

Model

R2

k

n

Zero order

0.953

0.301

-

First order

0.927

0.002

-

Higuchi model

0.988

5.168

-

Korsmeyer-Peppas model

0.972

-

0.517

 

Stability study:

No significant change was observed in physical and chemical properties of optimized formulation (F8) after 6 months of stability studies. However, relatively less change was observed in assay and release profiles of optimized formulation during its stability studies at different temperatures and relative humidity.


 

Table: 6 Results of stability studies of Tenofovir optimised formulation F8 before and after storage during the stability studies.

Formulation code

Parameters

Percentage of Drug content

Limits as per specifications

Initial

After 1st Month

After 3rd Month

After 6thMonth

F8

25˚C/60%RH % Release

97.67

97.14

96.85

96.23

Not less than 85%

F8

30˚C/75%RH % Release

98.86

98.26

97.78

96.74

Not less than 85%

F8

40˚C/75%RH % Release

98.89

98.45

97.62

97.21

Not less than 85%

F8

25˚C/60%RH Assay value

98.46

98.12

97.56

97.14

Not less than 90%

Not more than 110%

F8

30˚C/75%RH Assay value

98.64

98.23

97.84

97.43

Not less than 90%

Not more than 110%

F8

40˚C/75%RH Assay value

98.78

98.42

97.76

97.14

Not less than 90%

Not more than 110%

 


Fig: 11. Release profiles of Tenofovir optimised formulation F8 during the stability studies.

 

CONCLUSION:

In this current research work, TDF nanoparticles were prepared by nanoprecipitation method. It is observed that the concentration of PLGA played an important role in the entrapment and drug loading capacity of the formulation. On the basis of amount of drug loaded and entrapment efficiency the F8 formulation was considered as an optimized formulation and which is subjected to in vitro and stability studies. The compatibility study reveals that there is no incompatibility between the pure drug and optimized formulation (F8). The optimized formulation follows Higuchi release kinetics.

 

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Received on 29.04.2021           Modified on 16.07.2021

Accepted on 24.09.2021         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(7):3075-3080.

DOI: 10.52711/0974-360X.2022.00514