Formulation, Characterization and In-vitro Evaluation of Prolonged Local Drug Delivery of Antimicrobial on Bone Tissue Formation

 

Bandana Sharma1*, Chidambaram Soundrapandian2, Sonam Bhutia3

1,3Government Pharmacy College Sajong, Government of Sikkim, Sikkim University,

Sajong, Rumtek, East Sikkim, India – 737135.

2College of Health Science, Debre Tabor University, P.O. Box 272, Debre Tabor, Ethiopia.

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

 

ABSTRACT:

The present study aim to prepare and characterize prolonged local drug delivery of antimicrobial on bones tissue formation and its In-vitro evaluation. The results of drug released pattern were analysed after coating. In formulation (F1), the drug release was steep till 73%, where coating showed no such retardation effect. After 73% of drug release, F1 showed prolonged release of drug even after coating with 1% and 2% Chitosan, F2 and F3 showed prolonged better effect with 1% as compared to 2% Chitosan coating. In formulations containing one part of bioglass (F4 and F6) Showed prolonged release by 2% coating followed by 1% Chitosan coating. F5 was the exception where coating favoured dissolution. F8 and F9 showed prolonged release of drug by 1% than 2% Chitosan. In-case of In-vitro study, concentration was maintained above MIC i,e above 0.032 except in 816 hours where the drug concentration was observed below MIC (Fig No-14). F4C was found to be the best i,e 0.880 (Table No-06) for prolonged drug release over 6 weeks. FTIR study confirmed that characteristic peaks showed by Moxifloxicin (Fig No-3 & Table No-5) were 719cm-1, 1045cm-1, 1702cm-1 and 3327cm-1 which were also shown by developed formulations (725cm-1, 1051cm-1, 1703cm-1 and 3543cm-1). For In-vitro bioactivity, SEM were also performed after 24hrs (Fig No-15) and 48hrs (Fig No-16), the intensity of the HAP in 48hrs was more as compared to 24rs which confirmed that formulation is able to induced new bone cell after implantation. This result indicates that these newly prepared formulations could be a potential drug delivery system in osteomylietis condition (induces new bone cell) and can be helpful for the scientists for the particular field of study in near future.

 

KEYWORDS: Prolonged Local drug delivery, Osteomyelitis, Antimicrobial, bone formation, Characterization, Chitosan polymer.

 

 


INTRODUCTION: 

Osteomyelitis is inflammation of the bone caused by an infecting organism. Although bone is normally resistant to bacterial colonization, events such as trauma, surgery, presence of foreign bodies, or prostheses may disrupt bony integrity and lead to the onset of bone infection. Osteomyelitis can also result from hematogenous spread after bacteremia. Early and specific treatment is important in osteomyelitis, and identification of the causative microorganisms is essential for antibiotic therapy1.

 

 

All around 75% of cases of chronic osteomyelitis are caused by Staphylococcus aureus and coagulase-negative staphylococci2. Over 1.5 million osteoporotic fractures are reported annually in the USA alone, costing approximately $15 billion each year3, simultaneously the number of medications to treat and even prevent these diseases has expanded significantly in recent years4. The overall incidence of osteomyelitis is higher in developing countries. Outcome has not been overwhelming as with other disease conditions. Bone and soft tissue infections are serious problems in orthopaedic and reconstructive Surgery from which chronic osteomyelitis is a difficult infection to treat and eradicate. Long term parenteral antibiotics with multiple surgical debridements are often required for effective therapy5-6. Antibiotic treatment may be inadequate or ineffective in patients with poorly vascularised infected tissues and osteonecrosis, which is often present in cases of osteomyelitis. Moreover, normal doses of systemic antibiotics may be insufficient to rupture the biofilm produced by the infecting bacteria7. Moreover, insufficiency in local blood supply due to posttraumatic or post-operative tissue damage as well as inadequate tissue penetration or bacterial resistance decrease efficacy of systemic antibiotic therapy, both in terms of preventive or curative drug administration8-9. Even parenteral route experiencing poor supply of drugs at the site of treatment because of poor perfusion of bone. Higher systemic dose may lead to adverse effect.  In such situation local delivery of drugs offer various possibilities for avoiding serious side effects, avoiding infusions, decreased hospitalization, reduced medical expenses, release drug in a sustained fashion, maintain high drug concentrations locally, reduce presence of drug in systemic blood circulation, maintain drug stability for a longer period. Local release of drugs improved efficacy and cause faster healing. Inability of conventional therapy to access bone to maintain optimum drug concentration for required period, local drug delivery to one had been tried by modern medicine since 1970s10-13.

 

MATERIAL AND METHODS:

Standard antimicrobial drug: Moxifloxacin hydrochloride, purchased from Sigma Aldrich. Selected polymer: Chitosan, purchased from Yarrow Chem. Products, Mumbai (AR), OSTEOSET®, United States Gypsum Corporation.

 

Preparation of glass:

Glass was prepared by simple fusion method14.

 

Preparation of phosphate buffer saline:

Sodium chloride (NaCl)-8gm, Potassium chloride (KCl)-0.20gm, Di-sodium hydrogen phosphate-1.44gm, potassium di-hydrogen phosphate-0.24gm, and was taken it in a 100ml clean and dry volumetric flask and make up the volume up to 1000ml summarised in table No:01. Preparation of sample: A  stock solution of was prepared by taking 10mg of active drug and make up the volume up to 100ml with phosphate buffer saline. Concentration of the solution was calculated and found to be 100g/ml. The dilution was made to give the solution of 0, 0.5, 2, 4, 6, 8, 10(µg/ml) then the absorbance was taken at λmax of 287as given in diagram. With the given ratio of different ingredients, beads were prepared using distilled water as a solvent (Table No:02).  It was kept in desiccators for drying at room temperature until it dries completely (Fig No:01).

 

Coating of beads:

Coating of beads with 1% Chitosan solution and Coating of beads with 2% Chitosan solution are summarised in the following tables (Table No:03 and Table No:04).

 

Characterization:

Fourier transforms infrared radiation (FTIR) spectroscopy:

Drug and excipients interaction plays a vital role with respect to release of drug from the formulation amongst other15-18. FTIR technique has been used here to study the physical and chemical interaction between drug and excipients used. FTIR spectra were obtained using a FT-IR Spectrometer (Cari-630) of Agilent technology. The sample was scanned over a wave number in the region of 4000-400cm-1. The characteristic peaks were recorded for different samples.

 

In-vitro drug release studies: After complete drying of the beads each was placed in the clean tarson tube containing 5ml of phosphate buffer saline 7.4 and kept in incubator at 37ºC. Every 24 hours the phosphate buffer saline 7.4 was withdrawn and replaced with fresh phosphate buffer saline 7.4. The withdrawn phosphate buffer was filtered and from the filtrate 1ml was withdrawn and was diluted to check the absorbance from which drug release was calculated19. In-vitro drug release kinetic studies: The drug release data were subjected to release kinetic study (Fig No:02). Drug dissolution from solid dosage has been described by kinetic model in which the dissolved amount of drug (Q) is compared to the function of time (t). Some analytical definition of the Q versus t commonly used are, Zero order, First order, Higuchi and Korsemeyer-Peppas kinetic models20-25.

 

In-vitro Bioactivity study:

Sample was subjected to freshly prepared SBF having pH of 7.2 to 7.4 for 24 hours and 48 hours respectively. After 24 and 48 hours the sample was removed from the SBF solution and was washed properly with distilled water and dried properly. The dried sample was subjected to SEM analysis for study of surface morphology26-30.

 

RESULTS:

Standard graph of drug:

Table No: 01. Standard curve data of Moxifloxacin in phosphate buffer saline pH 7.4.

Serial  No.

Concentration in (µg/ml)

Absorbance

0.5

0.051

2

0.186

4

0.367

6

0.553

8

0.725

10

0.906

15

1.336

20

1.774

 

Fig:01: Standard curve of moxifloxacin HCl in phosphate buffer saline pH 7.4

 

Table No-02.Formulation design of beads in the ratio value without Chitosan coating.

Formulation

Drug

Glass

Osteoset®

F1

1

-NO-

5

F2

1

-NO-

7

F3

1

-NO-

9

F4

1

1

4

F5

1

1

6

F6

1

1

8

F7

1

2

3

F8

1

2

5

F9

1

2

7

 

Table No-03: Coating of beads with 1% Chitosan solution:

Formulation

Drug

(Moxifloxacin HCL)

Glass

(Bioactive glass)

Osteoset®

Coating with 1% Chitosan

F1C

1

-NO-

5

yes

F2C

1

-NO-

7

yes

F3C

1

-NO-

9

yes

F4C

1

1

4

yes

F5C

1

1

6

yes

F6C

1

1

8

yes

F7C

1

2

3

yes

F8C

1

2

5

yes

F9C

1

2

7

yes

 

Table No-04: Coating of beads with 2% Chitosan solution:

Formulation

Drug

(Moxifloxacin HCL)

Glass

(Bioactive glass)

Osteoset®

Coating with 2% Chitosan

F1D

1

-NO-

5

yes

F2D

1

-NO-

7

yes

F3D

1

-NO-

9

yes

F4D

1

1

4

yes

F5D

1

1

6

yes

F6D

1

1

8

yes

F7D

1

2

3

yes

F8D

1

2

5

yes

F9D

1

2

7

yes

 

 


 

 

Fig:02- Coating of beads with Chitosan different concentrations.

Characterization:

 

Fig:03: Fourier transforms infrared radiation (FTIR) spectroscopy of Moxifloxacin:

 

Fig:04: Fourier transforms infrared radiation (FTIR) spectroscopy of Formulation Beads:


 

Table No-05: Peaks and Ranges

Peaks shown by moxifloxacin

Peaks shown by formulation

Ranges

719cm-1

725cm-1

Benzene

700cm-1 – 750 cm-1

1045cm-1

1051cm-1

Heterocyclic stretching of N-H 1600cm-1 – 1300cm-1

1702cm-1

1703cm-1

Aliphatic carboxylic acid

1700cm-1 – 1690cm-1

3327cm-1

3543cm-1

Secondary amines ˃3000cm-1

 


In – vitro release profile of F1and F4

 

 

In – vitro release profile of F2,F5 and F8

 

In – vitro release profile of F3,F6 and F9

 

 

In – vitro release profile of F3, F6 and F9


Fig: 05: In-vitro release profile of F1, F2, F3, F4, F5, F6, F7, F8 and F9:



Table No-06: Kinetics models describe drug release from the prepared formulation:

Formulations

 

 Zero order  R2

First order R2

Higuchi

R2

Korsmeyer-peppas

Mechanism of dissolution

R2

n

F2

0.485

0.759

0.737

0.742

0.45

Non –Fickian or anomalous transport.

F3

0.508

0.762

0.758

0.750

0.45

Non –Fickian or anomalous transport.

F5

0.455

0.706

0.712

0.749

0.45

Non –Fickian or anomalous transport.

F6

0.454

0.720

0.706

0.702

0.43

Fickian diffusion

F2C

0.498

0.725

0.750

0.850

0.52

Non –Fickian or anomalous transport.

F3C

0.568

0.778

0.812

0.861

0.51

Non –Fickian or anomalous transport.

F4C

0.527

0.701

0.778

0.880

0.55

Non –Fickian or anomalous transport.

F6C

0.505

0.729

0.757

0.768

0.46

Non –Fickian or anomalous transport.

F8C

0.470

0.577

0.729

0.819

0.48

Non –Fickian or anomalous transport.

F9C

0.505

0.652

0.757

0.837

0.49

Non –Fickian or anomalous transport.

F2D

0.526

0.812

0.773

0.803

0.48

Non –Fickian or anomalous transport.

F3D

0.584

0.879

0.819

0.825

0.49

Non –Fickian or anomalous transport.

F4D

0.543

0.820

0.788

0.832

0.50

Non –Fickian or anomalous transport.

F6D

0.517

0.688

0.764

0.800

0.47

Non –Fickian or anomalous transport.

F8D

0.502

0.663

0.753

0.784

0.47

Non –Fickian or anomalous transport.

F9D

0.53

0.708

0.775

0.791

0.46

Non –Fickian or anomalous transport.

 


Kinetics models describe drug release from the prepared formulation. Thus the regression coefficient (R2) values was calculated for Zero order, First order, Higuchi and Korsmyer Peppas Model for the formulations able to prolonged the drug release over 6 weeks.

 

In-vitro Bioactivity study:

 

Fig: 06 SEM images of formulation after 24 hours and 48 hours of immersion in SBF:

 

DISCUSSION:

In-vitro release profile of the prepared formulation was studied. It was noticed that formulation containing appropriate amount of bioglass as well as Chitosan coating is required for prolonged release. In-vitro drug concentration in µg/ml was calculated and was observed that the drug concentration was maintained above MIC i.e. above 0.032 except in 816 hours where the drug concentration was observed below MIC. This might be the result of human error because the formulation was mixed and prepared by hand. The drug concentration in 360hrs and 456hrs showed a high concentration due to irregular sampling which justified that the dissolution medium was sufficient enough to overcome saturation. The drug release was depending on the inorganic in formulation F2, F3, F5 and F6. Though F5 and F6 have one part of bioglass substituted for OSTEOSET® which has reason to believe that it was the total ratio of the inorganic that was influencing the kinetics of drug release. This is justified by the similarity in the ratio of inorganic present in formulation F2 and F5 and in F3 and F6. Irrespective of the ratio of inorganic, when the beads are coated the retarding effect of inorganics has been overtaken by the coating polymer. An exponent value of 0.43 or less and 0.85 or above indicate respectively Fickian diffusion and case II transport (typical zero-order release). Values between 0.4 and 0.85 indicate non-Fickian or anomalous (by both diffusion and erosion) release. The release exponent (n) was calculated which showed F6 followed Fickian diffusion and majority of the formulation followed non-Fickian or anomalous (by both diffusion and erosion) transport. Both the inorganics have erodible properties which enhanced the dissolution mechanism by diffusion and erosion. SEM images (Fig.06) show the surfaces of the formulation after immersion in SBF after 24 hours and 48 hours respectively. This morphology is typical of HA-like material formed by the conversion of bioactive glasses in SBF solution. The intensity of the HAp in 48 hours was more compared to 24 hours which indicate that formulation is able to induce new bone cell after implantation.

 

CONCLUSION:

The formulations for prolonged local drug delivery of Moxifloxicin on bones were formulated by employing bioactive glass and osteoset as retardants. In-Vitro activity and instrumentation characterisation using FTIR and SEM confirmed that the formulations were bioactive. Among the formulations F4C was found to be the best formulation with R2 value of 0.880 followed according to KORSNEYER-PEPPAS Model. Hence, this result indicates that this newly prepared formulation could be a potential drug delivery system in osteomylietis condition and can be helpful for the research scientists for this particular field of study in near future.

 

ACKNOWLEDGEMENT:

The authors are grateful to the host institutions-Himalayan Pharmacy Institute, Majhitar, Government Pharmacy College Sajong-Sikkim-India and College of Health Science, Debre Tabor University, Ethiopia for providing with advanced facilities such as laboratory, chemical utilities, sophisticated instruments, man powder, sound and friendly environment, etc., to collect the significant interpretational data for this investigational work. Thanks to all the college team members especially to Dr.C.Soundrapandian and Mr. S. Bhutia for their kind guidance to complete this research work.

 

CONFLICT OF INTEREST:

The authors declared that there is no conflict of interest to disclose.

 

 

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Received on 22.03.2022             Modified on 16.08.2022

Accepted on 12.12.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1147-1152.

DOI: 10.52711/0974-360X.2023.00191