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  • Development And Evaluation of Proliposome Powder for Enhanced Delivery of Deflazacort
  • 1Dr. Vedprakash Patil Pharmacy College, Gevrai Tanda, Paithan Rd, Aurangabad,
    Maharashtra (INDIA).
    2Dr. Babasaheb Ambedkar Technological University, Lonere, Raigad
     

Abstract

This research thesis investigates the development and evaluation of Proliposome powder as a novel drug delivery system for enhancing the delivery of Deflazacort, a synthetic glucocorticoid with potent anti-inflammatory and immunosuppressive properties. Deflazacort is used in the management of various inflammatory and autoimmune conditions, including rheumatoid arthritis, asthma, and inflammatory bowel disease. However, its clinical utility is limited by poor aqueous solubility, low bioavailability, and systemic side effects. The formulation development phase involves the preparation of Proliposome by solvent evaporation technique, various lipid excipients and surfactants are screened to optimize the encapsulation efficiency, stability, and release characteristics of the Proliposome formulation. Total six batches were formulated from that batch 4 was giving the maximum results so selected as optimized batch. The evaluation of the formulated proliposome powder encompasses a comprehensive assessment of its physicochemical properties, including particle size, morphology, drug loading, and encapsulation efficiency. The entrapment efficiency was found in the range of 84.64 ± 0.5 to 91.01 ± 06. In vitro dissolution studies are conducted to investigate the release profile of Deflazacort from the proliposome powder, simulating physiological conditions encountered upon administration. The results were found in the range of 83.48% to 93.60%. The research findings contribute to the development of a novel drug delivery platform for Deflazacort, offering improved solubility, bioavailability, and therapeutic efficacy. The developed formulation has the potential to enhance patient compliance and therapeutic outcomes in the treatment of inflammatory and autoimmune disorders requiring glucocorticoid therapy.

Keywords

Deflazacort, Proliposomes, Glucocorticoid, autoimmune disorders, Bioavailability.

Introduction

Deflazacort (DFC) is a synthetic glucocorticoid widely prescribed for the management of various inflammatory and autoimmune conditions, including its prominent use as a first-in-class medication for the treatment of Duchenne Muscular Dystrophy (DMD), as recognized by the U.S. Food and Drug Administration (FDA).1-3 Despite its clinical significance, deflazacort faces significant limitations in its therapeutic application due to its poor aqueous solubility and consequently low bioavailability. These pharmacokinetic drawbacks hinder its absorption and overall therapeutic efficacy, necessitating the development of innovative drug delivery systems to overcome these challenges.4-5 Proliposomes, a novel lipid-based carrier system, have emerged as a promising approach to enhance the solubility and bioavailability of poorly water-soluble drugs like deflazacort. Unlike conventional liposomes, proliposomes are dry, free-flowing powders that, upon hydration, can form liposomes capable of encapsulating lipophilic drugs. This unique characteristic allows proliposomes to offer improved stability, increased drug loading capacity, and controlled release profiles, thereby enhancing the therapeutic effectiveness of poorly soluble compounds. Given their potential, proliposomes are particularly well-suited for the delivery of corticosteroids such as deflazacort, where optimizing drug release and absorption is critical for maximizing clinical outcomes.6-8 While proliposomes have been explored in various contexts for drug delivery, their application in enhancing the bioavailability of deflazacort remains unexplored. As of now, there has been no focused research investigating the development of proliposome-based formulations for this specific drug. Therefore, this study is pioneering in its approach, aiming to fill this gap in the literature by formulating proliposome powders for deflazacort to improve its bioavailability, stability, and overall therapeutic efficacy.9-12

The ultimate goal of this research is to develop a proliposome powder formulation that not only addresses the solubility and bioavailability issues associated with deflazacort but also provides a more reliable and controlled means of drug delivery. By optimizing the formulation, evaluating its physicochemical properties, and conducting in vitro release studies, this research seeks to establish a novel and effective drug delivery system. The successful development of such a formulation could significantly improve the therapeutic outcomes of patients, particularly those suffering from conditions like Duchenne Muscular Dystrophy, by ensuring better drug absorption and efficacy.13-15 Through this innovative approach, this study contributes to the broader field of drug delivery systems for poorly water-soluble drugs, advancing the possibilities of using proliposomes for enhancing the clinical performance of corticosteroids and other lipophilic drugs.16,17

MATERIALS AND METHODS:

MATERIALS:

Deflazacort used in the current study was generously provided as a gift sample by Anuh Pharma Ltd., Mumbai. A range of chemicals and reagents were utilized throughout the formulation and evaluation stages. Phosphatidyl Choline (Soya lecithin) was sourced from Himedia, while Cholesterol (AR grade) was procured from Finar Reagents. Mannitol (LR grade) was supplied by Sd Fine Chemicals Ltd. Methanol (AR grade) and Chloroform (AR grade) were obtained from Fisher Scientific, with Acetonitrile (AR grade) coming from Central Drug House Pvt. Ltd. In addition, Potassium Dihydrogen Orthophosphate (AR grade) was sourced from Sd Fine-Chem Ltd., and Sodium Hydroxide (LR grade) was supplied by Molychem. These chemicals and reagents were selected based on their quality and suitability for the study’s requirements.

Construction of calibration curve for Deflazacort

The solutions were prepared by weighing suitable quantity of Deflazacort drug on aluminum foil using pre-calibrated analytical weighing balance. The weighed quantity was transferred to volumetric flask and solubilized using purified Water. Prior to analysis, both cuvettes were washed with distilled water twice and rinsed with methanol twice to ensure complete cleaning. Then cuvettes were again rinsed with water .During UV analysis, both cuvettes were filled with water and reading was adjusted using Auto Zero button. Then, subsequent absorbance measurements were carried out.18

Preparation of Stock Solution

50 mg of Deflazacort was exactly weighed using pre-calibrated analytical balance, transferred in 50 mL volumetric flask and dissolved in sufficient water using sonication. It produced a solution of 1mg/mL strength (Stock-1). The Stock-1 containing flask was covered with foil and sealed with paraffin film to avoid degradation and loss due to evaporation.18

Determination of Analytical Wavelength (?max)

Sufficient volume of Stock-1 was scanned under UV region of 400-200 nm using water as blank. The wavelength, at which there was maximum absorption, was selected as wavelength for analysis.

Preparation of Standard curve

A five point standard curve of Deflazacort was prepared using different concentrations of drug from Stock-1 solution. The concentration range selected was from 20µg/mL to 100µg/mL. Concentrations of 20µg/mL, 40µg/mL, 60µg/mL, 80µg/mL, and 100µg/mL were prepared by appropriate dilutions of Stock-1 solution.

Formulation of Preliminary trial batches


Table 1: Quantity of chemicals required for formulation of Proliposomes

Sr. No.

Chemicals

Quantity taken

1

Posphatidyl choline

7 mg

2

Cholesterol

5 mg

3

Drug

12 mg

4

Mannitol

600mg

5

Solvents (Cloroform: Methanol)

20ml (18:2)


Evaluation test for Preliminary trial batches

Microscopy

A drop of distilled water was added to Proliposome powder on glass slide without cover slip. It was observed for formation of liposome from proliposome formulation under a microscope.

Entrapment Efficiency

The entrapment efficiency of proliposome formulation was determined by dissolving Proliposome powder equivalent to 2000µg of drug in 1ml of Acetonitrile. It was vortexed for 30 sec. and then centrifuged at 10,000 rpm for 15 min at RT. The 500 µl supernatant liquid was collected and transferred into pre-labeled 5ml volumetric flask. The volume was made up to 5ml using Acetonitrile and analyzed with UV spectrophotometer at 266 nm.18

Formulation Development19,20

Method of preparation of Proliposome formulation

The Proliposomes of Deflazacort were prepared by Solvent Evaporation Technique. Proliposomes were prepared in a Round Bottom Flask (RBF).Chloroform and Methanol was used as a solvents and Mannitol as a carrier.


Table 2: Total batch size of Proliposome Batches

Batch No.

Phosphatidylcholine (mg)

Cholesterol (mg)

Drug (mg)

Mannitol(mg)

Methanol (mL)

Chloroform (mL)

B1

7

5

12

600

18

2

B2

5

7

12

600

18

2

B3

3

9

12

600

18

2

B4

9

3

12

600

18

2

B5

8

4

12

600

18

2

B6

4

8

12

600

18

2


Following steps were followed during formulation of Proliposomes

  1. Weighing of Drug and Lipids

Drug and Lipids were accurately weighed on aluminum foil using pre-calibrated analytical balance.

  1. Preparation of solvent mixture

Both organic solvents were measured accurately and mixed in beaker. This solvent mixture was used to dissolve Drug and Lipids.

  1. Preparation of Lipid mixture

Accurately weighed amounts of Lipids were dissolved in sufficient solvent mixture, separately. The solutions of two Lipids were mixed in a RBF. The RBF was sealed with aluminum foil to avoid loss of solvent mixture by evaporation.

  1. Loading of Drug

Accurately weighed Drug was dissolved in sufficient solvent mixture. This solution was transferred carefully and completely to the RBF containing lipid mixture.

  1. Addition of Excipient

The Excipient material (Mannitol) was weighed accurately using pre-calibrated weighing balance on an aluminum foil. It was carefully transferred to the RBF containing Drug and lipids. Remaining solvent mixture was added to it with washing of aluminum foil.

  1. Preparation of Proliposomes

The RBF containing lipids, drug and carrier was shaken by hand for complete mixing of chemicals. All material got dissolved in solvent mixture, except carrier material; hence resulting in a suspension of mannitol. This flask was attached to a rotary vacuum evaporator and the organic solvents from this mixture were evaporated under reduced pressure at the temperature 40°±2°C. After ensuring the complete removal of solvents, the resultant powder was further dried in a vacuum desecrator at room temperature so as to obtain dry, free flowing powder. This powder was stored intightly closed container at 4°C for further evaluation.Diagrammatic Representation and Proliposome powder layer formed by Solvent Evaporation Technique were given in Figure 1.

       
            Diagrammatic representation of Solvent Evaporation Technique.png
       

Figure 1: Diagrammatic representation of Solvent Evaporation Technique

Evaluation of Proliposome Formulation21-23

Flow Properties

The flow properties of proliposome powders were assessed through measuring the angle of repose, Carr’s compressibility index and Hausner’s ratio. The angle of repose was determined by using conventional fixed funnel method. A glass funnel was usedwith an orifice of 10 mm. The height of funnel from the beginning to end of orifice was 111 mm. The funnel was fixed in place, 4 cm above the bench surface. The height (h) of the pile formed by the cone of powder and the radius (r) of the base were measured. The Carr’s compressibility index and Hausner’s ratio were calculated from the bulk and tapped density of the proliposome powders. Bulk and tapped densities were determined using the methods outlined in the USP. Sufficient proliposome powder was passed through a mesh size #18 sieve into a pre-weighed 25 ml graduated cylinder with 0.5 ml markings. The bulk volume was measured after manually tapping the cylinder two times on a flat table top surface. The tapped volume was measured with the tap density tester (OSWORLD JRIC-36).

Measurement of Vesicle size and Number of Vesicles/mg of Proliposome powder

1 mg of proliposome powder was accurately weighed. A drop of distilled water was added to this powder on glass slide without cover slip. It was observed for formation of liposome from proliposome formulation. Vesicle size and count was recorded under Digital optical microscope [DMWBI-223ASC, motic] with magnification 10x.

Fourier Transform Infrared Spectroscopy (FT-IR)

After starting the instrument, ‘Background Measurement’ was performed without placing the drug on panel. For ‘Sample Measurement’, pure Deflazacort was placed on cleaned panel of FT-IR Spectrometer (Bruker). The placed Deflazacort was sandwiched between panel and upper arm. This sample was scanned over a wave number range of 4000 to 400 cm-1. Similar procedure was followed for Deflazacort, Phosphatidyl choline, Cholesterol, Mannitol, blank proliposomes.

Drug content Test

The Deflazacort content was determined by dissolving the Proliposome powder equivalent to the 2000 µg of drug in 1ml of Methanol. The aliquot sample was taken in to microcentrifuge tube and vortexed for 30 sec. The micro-centrifuge tube was sonicated for 2 min. ensuring that all lumps are broken. It was centrifuged at 10,000 rpm for 15 min at RT. The 500 µl of supernatant liquid was collected and transferred in to labeled 5ml volumetric flask. The volume was made up to 5ml using Methanol and analysed with UV spectrophotometer at wavelength of 266 nm.

Entrapment Efficiency

The entrapment efficiency of proliposome formulation was determined by dissolving Proliposome powder equivalent to 2000 µg of drug in 1ml of Acetonitrile. It was vortexed for 30 sec. and then centrifuged at 10,000 rpm for 15 min at RT. The 500µl supernatant liquid was collected and transferred into labeled 5ml volumetric flask. The volume was made up to 5ml using Acetonitrile and analyzed with UV spectrophotometer at 266 nm.

In-Vitro Dissolution study

In-vitro dissolution study of Deflazacort Proliposome was performed using USP type II Dissolution testing apparatus fitted with paddles. The speed of rotation of paddle was set at 50 rpm. Dissolution study was carried out using 500ml of phosphate buffer (pH 7.4) maintained at a temp of 37°± 5°C under sink condition. Proliposome powder equivalent to 12 mg of drug was added to the dissolution medium. At a predetermined time intervals of 5, 10,15,30,45 min. for first hours and for next 24 hours samples were collected.  5 ml samples were withdrawn, filtered through Whatman filter paper, and analyzed by UV spectrophotometer at 266 nm. Drug content was determined by placing absorbance in a standard curve.

Stability study

Proliposome stability study was performed as per the method reported previously. The Proliposome formulation stored in glass vials were covered with aluminium foil and kept at room temperature and refrigerator at 40C for a period of 1 month. After 1 month samples were withdrawn and hydrated with distilled water and observed for any sign of drug crystallization under optical microscope and evaluated for color & Entrapment.

RESULTS AND DISCUSSION:

Construction of calibration curve for Deflazacort

Maximum wavelength detection for Deflazacort was found at 242 nm. The standard calibration curve of Deflazacort was obtained by plotting the absorbance of the standard solution against its concentration at 242 nm. The standard solution of Deflazacort showed the linear curve with correlation coefficient of 0.9993. Their equations of lines was y = 0.0091x + 0.0935 at selected ?max. Following table 3 shows absorbance of respective standard solution. The standard curve for Deflazacort at 242 nm.


Table 3: Calibration range for Deflazacort

Sr. No.

Concentration (µg/mL)

Absorbance

1

20

0.275

2

40

0.453

3

60

0.646

4

80

0.834

5

100

0.997


       
            Calibration curve for Deflazacort.png
       

Figure 2: Calibration curve for Deflazacort

Evaluation test for Preliminary batches

Microscopy

The microscopy test of Preliminary batch of Proliposomes was performed. Following images were obtained from digital microscope showing formation of Proliposomes vesicles. The Average size of vesicles was found to be 200-300 nm. Formation of vesicles after hydration with distilled water was shown in Figure 3.

       
            Formation of vesicles after hydration with distilled water (preliminary batch).jpg
       

Figure 3: Formation of vesicles after hydration with distilled water (preliminary batch)

Entrapment Efficiency

Preliminary batch of Proliposomes were prepared using formula reported in previous reports. Entrapment efficiency was found to be 76%. These tests suggest that the Proliposomes were formed with good entrapment efficiency.

Evaluation Test of designed batches

Flow properties

Flow properties are one of the best parameter to evaluate the Proliposome formulation. Three types of flow measurements can be used to evaluate the nature of powder flow which is angle of repose; Carr’s index and Hausner’s ratio. It is stated that, smaller the value of angle of repose (<30>


Table 4: Flow Properties of Proliposome Formulation

Batch Code

h/r

Angle of repose (?)

Bulk Density

Tapped Density

Carr's Index (%)

Hausner Ratio

B1

0.424

23

0.51

0.63

19.04

1.23

B2

0.700

35

0.58

0.69

15.94

1.18

B3

0.424

23

0.51

0.63

19.04

1.23

B4

0.700

14

0.59

0.68

13.23

1.15

B5

0.674

34

0.53

0.68

22.05

1.28

B6

0.674

34

0.54

0.69

21.73

1.27


In, present work it was observed that the  batch B1,B3 & B4  shows angle of repose less than 30°, Carr’s index and Hausner’s ratio also less than 21 & 1.25 respectively indicating the good flow Proliposome powder formulation. The Flow Properties of batches 1-6 was performed.

Measurement of Vesicle size and Number of Vesicles/mg of Proliposome powder

Optical microscopy was used as primary tool to determine the vesicle size of the prepared Proliposomes by observing them under 10x. The vesicle size determination can be done by hydrating the Proliposome powder followed by manual agitation and determining particle size using Digital Microscope. Distinctive advantage of Proliposome formulation is speculated only when abundant number of vesicles are derived from hydration of Proliposomes which form an important prerequisite for optimizing the composition of the same.


Table 5: Measurement of Vesicle size and Number of Vesicles/mg of Proliposome Formulation

Batch Code

Vesicle size Range (nm)

 

Total No. of Vesicle

 

50-100

100-200

200-300

 

1

4

18

154

176

 

2

4

19

61

84

 

3

4

17

149

200

 

4

10

46

77

133

 

5

7

43

167

217

 

6

5

12

83

100

 


       
            Formation of vesicles after hydration with distilled water.png
       

Figure 4: Formation of vesicles after hydration with distilled wate

The mean size of the vesicles was in the range of 180 to 290 nm as the concentration of cholesterol increases the vesicle size was also increases as shown in figure. In, present work it was observed that the vesicle size of all the batches are within the range of 50-300nm. Batch B1, B3, B4 & B5, had maximum concentration of cholesterol indicates the increase in the vesicle size of Proliposomes. Number of vesicles and vesicle size of batch 1-6 was measured by using optical microscope.

Fourier Transform Infrared Spectroscopy

From FTIR studies, the characteristic bands for important functional groups of Deflazacort, Phosphatidylcholine, Cholesterol, Mannitol, Blank Proliposome and Proliposome were identified. The spectrum of Deflazacort was recorded in a FTIR.

       
            FTIR Spectra of Deflazacort.png
       

Figure 5: FTIR Spectra of Deflazacort

       
            FTIR Spectra of Phosphatidylcholine.jpg
       

Figure 6: FTIR Spectra of Phosphatidylcholine

       
            FTIR Spectra of Cholesterol.jpg
       

Figure 7: FTIR Spectra of Cholesterol

       
            FTIR Spectra for Mannitol.jpg
       

Figure 8: FTIR Spectra for Mannitol

       
            FTIR Spectra of Blank Proliposome.jpg
       

Figure 9: FTIR Spectra of Blank Proliposome

       
            FTIR Spectra of Proliposome batch B4.jpg
       

Figure 10: FTIR Spectra of Proliposome batch B4

FTIR study of Proliposomes was done from this sudy it was observed that, there was no significant difference in FTIR spectrum of Deflazacort and Deflazacort loaded Proliposomes were observed when they were overlapped. The characteristic asymmetric and symmetric stretching vibrations of equivalent C-O stretching, C-H bending bands in Deflazacort which was unchanged in FTIR study of Proliposomes. These results were suggested that there is no chemical interaction between drug & lipids and drug stability during encapsulation process. Observed frequencies were appeared in the range of reported frequencies hence confirming the respective functional groups in the structure.

Drug Content Test

Proliposome also evaluated for % drug content from these it was observed that the total drug in the formulation was obtained to be in the range of 77.31% to 89.57%. The drug content test of batch 1-6 was performed.


Table 6: Drug Content Test

Batch Code

% Drug Content*

B1

82.87±0.2

B2

80.50±0.2

B3

77.31±0.3

B4

89.57±0.7

B5

78.79±0.3

B6

84.96±0.3


*All values are mean±SD (n=3)

Entrapment Efficiency test

Entrapment efficiency of Proliposome formulation was also carried out. The maximum benefit of proliposomes can be achieved when high entrapment efficiency after hydration in the gastric fluids occurs.


Table 7: Entrapment Efficiency Test

Batch Code

% Entrapment*

B1

84.64 ± 0.5

B2

84.72 ± 0.2

B3

86.23 ± 0.3

B4

91.01 ± 06

B5

86.53 ± 01

B6

88.77 ± 0.1


*All values are mean±SD (n=3)

The entrapment efficiency of proliposome formulations was obtained between 84 and 93%. It was observed that entrapment efficiency is dependent on the composition of Proliposomes. The entrapment efficiency increases with increase in the concentration of cholesterol and Phosphatidylcholine. This can be owed to the decreased leakage of the drug because of high compactness and hydrophobic interactions making the bilayer more stable. In present work batch B1, B6, & B4 had maximum concentration of phosphatidylcholine and cholesterol indicates the high entrapment efficiency. The Entrapment efficiency of batch 1-6 was performed.

In- vitro Dissolution Study

In-vitro drug release studies of the Proliposome powder were conducted for a period of 24 hours. In order to ascertain the effect of composition of proliposomes on the drug release in vitro release study was conducted.


Table 8: Percent (%) Drug release of batch B1-B6

 

Time (hr)

% Drug release

 

B1

B2

B3

B4

B5

B6

0

0

0

0

0

0

0

0.08

2.26

3.16

2.26

2.93

0.48

2.93

0.1

4.97

5.42

5.19

5.20

3.61

5.64

0.25

9.04

10.17

9.04

13.73

6.10

8.38

0.5

15.15

16.30

15.83

16.77

9.96

14.46

0.75

20.44

20.25

20.45

21.41

11.84

18.88

1

28.24

32.07

30.03

32.56

28.26

29.56

1.5

33.20

34.39

33.23

35.34

36.36

32.53

2

37.77

39.64

39.14

40.59

38.28

38.43

3

41.49

42.26

41.53

43.00

39.77

40.81

4

47.02

47.58

47.73

47.65

48.86

47.01

6

51.49

52.95

51.54

58.61

53.35

51.04

8

56.45

58.81

56.95

59.39

54.98

56.22

12

62.13

64.06

62.18

62.86

60.42

62.56

24

83.48

88.78

85.77

93.60

78.63

84.36


       
            Dissolution Profile of Batch B1-B6.png
       

Figure 11: Dissolution Profile of Batch B1-B6

The initial rapid rise in the release was observed could be due to the burst release of drug because of the presence of unentrapped drug in the outer region of liposomes. In present work batch B5, B4 & B6 shows higher entrapment indicates the well release. The percent drug release was obtained in the range of 78.63% to 93.60%. Table 8 shows % drug release of batch B1- batch B6 and were shown in figure 11.

Stability study

Proliposomal batch 4 (B4) was kept for stability study at 4oC and room temperature for one month observed for any Drug crystallization under microscope and evaluated for colour, and entrapment efficiency. Stability parameter of Proliposome formulation was given in Table 9. From the stability study of batch 4 it was observed that the physical appearance of the B4 did not change when sample were stored at 4°C for 1 month. It can be inferred from the observed data that the prepared Proliposome B4 was stable after one month of storage at 4 ± 1°C.


Table 9: Stability parameter of Proliposomes

Sr. No.

Storage condition

Drug crystallization

Color

Entrapment (%)

1

Initial

No

White

93.46

2

1 Month

No

White

91.12


CONCLUSION:

The research successfully developed and evaluated a proliposome powder formulation for the enhanced delivery of Deflazacort, addressing its challenges of poor aqueous solubility, low bioavailability, and systemic side effects. Six batches were formulated, and batch B4 was identified as the optimized formulation based on superior performance in encapsulation efficiency (91.01 ± 0.6%), drug content (89.57 ± 0.7%), and in-vitro drug release (93.60% over 24 hours). The proliposome formulation demonstrated excellent physicochemical properties, including good flow characteristics (angle of repose <30>

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest

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Reference

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Photo
Ashwini Khangan
Corresponding author

Dr. Vedprakash Patil Pharmacy College, Gevrai Tanda, Paithan Rd, Aurangabad, Maharashtra (INDIA).

Photo
Vaibhav Changediya
Co-author

Dr. Babasaheb Ambedkar Technological University, Lonere, Raigad

Ashwini Khangan*, Vaibhav Changediya, Development and Evaluation of Proliposome Powder for Enhanced Delivery of Deflazacort, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 12, 563-575. https://doi.org/10.5281/zenodo.14287973

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