Design, development and characterization of carrier mediated drug delivery system for effective management of Osteoarticular Tuberculosis

: Tuberculosis (TB) is a potentially fatal contagious disease and is a second leading infectious cause of death in world. Bone and joint TB (BJTB)/ Osteoarticular TB is a secondary form of TB occurring most commonly due to hematogenous seeding of mycobacterium (MTB) from the primary site of infection. The prevalence of the disease is around 30 million globally and approximately 30% or 10 million cases exist in India. Osteoarticular tuberculosis, is found in about 10–20% of all diagnosed tuberculosis, is the most common extrapulmonary tuberculosis. Osteoarticular TB is treated using standard regimen of 1stand 2ndline Anti- Tubercular drugs (ATDs) for extensive period of 8 - 20 months. These drugs are commonly administered in high doses by oral route or by intravenous route, because of their compromised bioavailability. The common drawbacks associated with conventional therapy are poor patient compliance due to long treatment period, frequent and high dosing and toxicity. Additionally failure by the patients to follow therapeutic regimen properly has given rise to development of resistance against these ATDs. This aspect marks for the need of formulations to eliminate these drawbacks. MTB is an intracellular pathogen of mononuclear phagocyte. This attribute makes nanotherapeutics an ideal approach for MTB treatment as macrophages capture nano forms. Polymeric nanoparticles are removed from the body by opsonization and phagocytosis, this forms an ideal strategy to target macrophage containing mycobacteria. To further improve targetability, the nanoparticles are conjugated with ligand, which serves as an easy substrate for the receptors present on the macrophage surface. The purpose of present work was to develop intra articular injectable in-situ gelling system containing polymeric nanoparticles, which would have promising advantages over conventional method of treatment. The rationale behind formulating nanoparticle incorporated in situ gel based system was to ensure localization of the formulation in intra articular cavity along with sustained release and conjugation of nanoparticles with mannose as ligand to improve uptake by macrophages. Rifampicin standard ATD was formulated into Chitosan nanoparticles. Chitosan with 85% degree of deacetylation (DDA) and Sodium tripolyphosphate (TPP) as the crosslinking agent was used for preparing nanoparticles. The percent entrapment was found to be about 71%. The prepared nanoparticles were conjugated with mannose. Conjugation of ligand was ascertained by performing Fourier transformed Infrared spectroscopy. The particle size was found to be in the range of 130 -140 nm and zeta potential of 38.5 mV. Additionally we performed scanning electron microscopy to characterize the surface morphology of ligand-conjugated nanoparticles. The conjugated Chitosan nanoparticles were incorporated into in situ gelling system comprising of Poloxamer 407 and HPMC K4M. The gelling system was evaluated for viscosity, gelling characteristics and syringeability. The drug release from conjugated nanoparticles incorporated in in situ gel was found to be about 70.3% at the end of 40 hours in simulated synovial fluid following zero order release kinetics. Based on the initial encouraging results obtained, the nanoparticles are being envisaged for ex-vivo cellular uptake study using TB infected macrophages


ABSTRACT:
Tuberculosis (TB) is a potentially fatal contagious disease and is a second leading infectious cause of death in world. Bone and joint TB (BJTB)/ Osteoarticular TB is a secondary form of TB occurring most commonly due to hematogenous seeding of mycobacterium (MTB) from the primary site of infection. The prevalence of the disease is around 30 million globally and approximately 30% or 10 million cases exist in India. Osteoarticular tuberculosis, is found in about 10-20% of all diagnosed tuberculosis, is the most common extrapulmonary tuberculosis. Osteoarticular TB is treated using standard regimen of 1 st and 2 nd line Anti-Tubercular drugs (ATDs) for extensive period of 8 -20 months. These drugs are commonly administered in high doses by oral route or by intravenous route, because of their compromised bioavailability. The common drawbacks associated with conventional therapy are poor patient compliance due to long treatment period, frequent and high dosing and toxicity. Additionally failure by the patients to follow therapeutic regimen properly has given rise to development of resistance against these ATDs. This aspect marks for the need of formulations to eliminate these drawbacks. MTB is an intracellular pathogen of mononuclear phagocyte. This attribute makes nanotherapeutics an ideal approach for MTB treatment as macrophages capture nano forms.
Polymeric nanoparticles are removed from the body by opsonization and phagocytosis, this forms an ideal strategy to target macrophage containing mycobacteria. To further improve targetability, the nanoparticles are conjugated with ligand, which serves as an easy substrate for the receptors present on the macrophage surface. The purpose of present work was to develop intra articular injectable insitu gelling system containing polymeric nanoparticles, which would have promising advantages over conventional method of treatment. The rationale behind formulating nanoparticle incorporated in situ gel based system was to ensure localization of the formulation in intra articular cavity along with sustained release and conjugation of nanoparticles with mannose as ligand to improve uptake by macrophages. Rifampicin standard ATD was formulated into Chitosan nanoparticles. Chitosan with 85% degree of deacetylation (DDA) and Sodium tripolyphosphate (TPP) as the crosslinking agent was used for preparing nanoparticles. The percent entrapment was found to be about 71%. The prepared nanoparticles were conjugated with mannose. Conjugation of ligand was ascertained by performing Fourier transformed Infrared spectroscopy. The particle size was found to be in the range of 130 -140 nm and zeta potential of 38.5 mV. Additionally we performed scanning electron microscopy to characterize the surface morphology of ligand-conjugated nanoparticles. The conjugated Chitosan nanoparticles were incorporated into in situ gelling system comprising of Poloxamer 407 and HPMC K4M. The gelling system was evaluated for viscosity, gelling characteristics and syringeability. The drug release from conjugated nanoparticles incorporated in in situ gel was found to be about 70.3% at the end of 40 hours in simulated synovial fluid following zero order release kinetics. Based on the initial encouraging results obtained, the nanoparticles are being envisaged for ex-vivo cellular uptake study using TB infected macrophages Keywords: Osteoarticular tuberculosis, Chitosan, mannose, ligand, conjugation Tuberculosis (TB) is a potentially fatal contagious disease and is a second leading infectious cause of death in world. Bone and joint TB (BJTB)/ Osteoarticular TB is a secondary form of TB occurring most commonly due to hematogenous seeding of mycobacterium (MTB) from the primary site of infection (1). The prevalence of the disease is around 30 million globally and approximately 30% or 10 million cases exist in India. Osteoarticular tuberculosis, is found in about 10-20% of all diagnosed tuberculosis, is the most common extrapulmonary tuberculosis. Osteoarticular disease is seen after the principal lesions of lungs spreads phenomenally via lymphnodes (2). Osteoarticular TB is treated using standard regimen of 1 st and 2 nd line Anti-Tubercular drugs (ATDs) for extensive period of 8 -20 months. The predisposing factors affecting success of therapy include patient compliance, therapy regimen, malnutrition, pre-existing diseases (3)(4). Osteoarticular TB behaves differently from pulmonary TB. The treatment is comparatively complex than pulmonary TB and requires combination therapy for longer duration around a year or two as destroying dormant population is quite challenging. Inadequate killing of MTB can cause relapse with variation in treatment response (5,6) The conventional therapy regimens have been successful to some extent in bringing about, if not complete but at least partial eradication of TB. The drawbacks associated with currently available treatment mainly include long period of treatment, toxicity, increased frequency of dosing (7). All these factors lead to discontinuation of the therapy even before the completion of treatment. The repercussions of discontinued therapy are increased probability of MTB bacteria developing resistance to drug leading to ineffectiveness of treatment (8). Most of the drugs used for the treatment of TB belong to BCS II or IV. It is because of their compromised solubility or permeability resulting into poor bioavailability. As a result they are required to be administered in high doses by the oral route or by intramuscular /intravenous route (9). This marks for the need of evolving and developing modified formulations to eliminate these drawbacks. Nanocarriers have the potential to address some of the challenges like an ideal approach for MTB treatment as macrophages capture nanoforms with or without surface modifications (10).Various nanocarrier systems that have been extensively studied are polymeric and lipid nanoparticles, polymeric micelle, carbon nanotubes (11). The peculiar characteristic of nanocarriers is that they are removed from the body by opsonization and phagocytosis. This attribute forms an effective strategy to target macrophage containing mycobacteria.
Although nano formulations can play an important role in effective treatment of TB, their role in enhancing targetability is sometimes limited. In order to improve the targetability of nanoformulations, various ligands specific to the mycobacteria cell components or macrophage receptor are being envisaged (12) .Ligand anchored therapy is a strong and effective approach to execute drug delivery into selective target cells.. Studies have shown that apart from targeting, ligands provide shielding effect to drug carrier and prolong its circulation time in the bloodstream (13). In the present work we have formulated Chitosan nanoparticles loaded with first line therapy drug Rifampicin. The polymeric nanoparticles were conjugated with mannose as targeting ligand. The conjugated drug loaded polymeric nanoparticles were incorporated into intra articular injectable insitu gelling system. The rationale behind developing this formulation strategy was to ensure localization of the formulation in intra articular cavity along with sustained release and conjugation of nanoparticles with mannose as ligand to improve uptake by macrophages. It has been observed that mannose receptors are over expressed during mycobacterial infection. Hence mannosylated nanoparticle can serve as effective cargo to deliver the drug in to macrophageal cells hosting the mycobacteria (14)

Materials and Method
The active pharmaceutical ingredient, Rifampicin, was obtained as a gift sample from Lupin Pharmaceuticals Ltd., India. The natural polymer, Chitosan with 85% degree of acetylation from were obtained as gift sample. The other chemicals and solvents required for experimentation were procured from SD Fine Chemicals Ltd.

Preparation of Rifampicin loaded Mannose conjugated Chitosan nanoparticles
In order to formulate Rifampicin loaded mannose conjugated chitosan nanoparticles, mannose conjugated Chitosan polymer was prepared. Mannose conjugation was obtained by performing reductive deamination of Chitosan with D-Mannose (15). Chitosan was dissolved in Lactic acid solution (1%w/v) using overhead stirrer. This was followed by addition of aqueous solution of STAB and D-Mannose to the chitosan solution. The polymer was allowed to react with STAB and D- Mannose for a period of 2 days. On completion of reaction, unreacted mannose and STAB were removed. The prepared polymer was dried using Spray Drier (Labultima LU 222) maintaining inlet temperature at 50°C (16). The process was optimized for parameters like % STAB concentration and % D-Mannose concentration. The formation of conjugated polymer was ascertained by Fourier Transformed Infrared Spectroscopy ( Figure 2D). Rifampicin , the model drug used for loading nanoparticles was subjected to preformulation studies wherein the purity of drug was assessed by performing Differential Scanning Calorimetry (DSC) and FTIR(Shimadzu IRAffinity-IS) (Figure 1 and 2A). Chitosan nanoparticles were produced by ionic gelation method using TPP as crosslinking agent (17). In order to prepare nanoparticles, the conjugated polymer was dissolved in 2% w/v Lactic acid solution under magnetic stirring for 2 hours (18)

Chitosan nanoparticles
Various gelling agents such as Chitosan, Deacetylated Gellan Gum, Sodium Alginate, Poloxamer 407 in combination with HPMC K4M were used to formulate in situ gelling system (23). The composition and proportion of the polymers were optimized based on clarity, syringeability, gelation time, gelling capacity. Rifampicin loaded mannose conjugated chitosan nanoparticles were incorporated in in situ gelling system and evaluated for drug release for period of 40 hours (24) 3. Results and Discussion:

Preparation of Rifampicin loaded Mannose conjugated Chitosan nanoparticles
Rifampicin is a bactericidal drug which interferes with the synthesis of nucleic acids by inhibiting DNA-dependent RNA-polymerase. Rifampicin binds to the pocket of RNA polymerase beta subunit within the DNA/RNA channel but away from the active site. The drug is active against Gram-positive bacteria, mycobacterium species and some Gram-negative bacteria (25).The drug shows good pH dependent solubility at lysosomal pH of the macrophage. By conventional route Rifampicin is required to be administered in high and frequent doses in order to exhibit therapeutic effect. In order to counter and nullify some of these drawbacks we have formulated intraarticular injectable in-situ gelling system containing polymeric nanoparticles. The objective behind formulating nanoparticle incorporated in situ gel based system is to ensure localization of the formulation in intra articular cavity along with sustained release and easy access of nanoparticles to bone and joints thus, minimizing bio distribution to other parts. The drug was evaluated for preformulation testing to ascertain its purity and compliance with compendial standards. An endothermic peak assignable to melting point of Rifampicin was observed at 183 -185°C indicating purity of active pharmaceutical ingredient (Figure 1). FTIR peaks were observed at wave numbers peculiar to the functional groups present in molecular structure of Rifampicin further confirmed the suitability and quality of the active pharmaceutical ingredient (Table   1 and Figure 2A).   In the pre-optimization experiments Chitosan nanoparticles were prepared during polymer of different DDA. The objective of performing these experimental trials was to select the suitable grade of Chitosan, for preparing conjugated polymer. Polymeric nanoparticles were prepared using Chitosan 75% and Chitosan 85% respectively along with TPP 1% as crosslinker (Table 2). Nanoparticles prepared using 75% and 85% DDA were subjected to microscopic observation using Motic. Chitosan with 75% DDA yielded thread like aggregates and instant gelation of particles was observed.
Nanoparticles obtained from Chitosan with 85% DDA were found to be spherical ( Figure 3)   (Table 3). Mannose conjugated Chitosan polymer prepared by spray drying when freshly prepared was found to be off white and free flowing powder. However as the time elapsed, a drastic change in colour from off white to brown was observed for conjugated polymer. The time required for this colour change was found to be dependent on mannose and STAB proportion. Batch CM5 with reduced concentration of STAB and Chitosan and mannose in the ratio 3:1 gave stable free flowing powder. The conjugation of mannose with Chitosan was ascertained by overlaying the FTIR spectra of Chitosan and mannose with that of conjugated polymer (Table 4 and Figure 2C). In the

Evaluation of Rifampicin loaded mannose conjugated chitosan nanoparticles
Rifampicin loaded mannose-conjugated chitosan nanoparticles of Batch M8 were assessed for physicochemical characteristics. For comparative evaluation, nanoparticles were prepared using plain chitosan. In order to determine entrapment efficiency, 10ml of the dispersion was centrifuged at15, 000 RPM at4°C for 20 minutes. The supernatant was collected, diluted with methanol and analyzed for free drug. Entrapment Efficiency was determined by using the following formula: (26) %Entrapment Efficiency = for plain Chitosan nanoparticles whereas for mannose conjugated Chitosan nanoparticles, the size was observed to be 142 nm with PDI of 0.154 ( Figure 4A and 4B) . It was concluded that the coupling of mannose in chitosan caused slight increase in the size of nanoparticles prepared using conjugated polymer. Zeta potential of Chitosan particles was found to be 42.6 mV whereas zeta potential of mannose conjugated Chitosan particles was found to be 38.5 mV. The reduction in zeta potential was attributed to conjugation of mannose to Chitosan resulting in reduction of primary amino groups as they couple to CHO groups of Mannose ( Figure 4C) (27).
Surface morphology of mannose-conjugated chitosan nanoparticles and chitosan nanoparticles was compared by performing Scanning Electron Microscopy (SEM) ( Figure 5). The formulations were placed on circular aluminum stubs using double adhesive tape, coated with Gold in HUS-5 GB vacuum evaporator, and observed in SEM at an acceleration voltage of 10 kV and a magnification of 5000 X (28). SEM images revealed that plain chitosan nanoparticles were spherical whereas mannose conjugated chitosan nanoparticles were found to be platelet shaped with pitted surface. This peculiar characteristic was attributed because of conjugation of mannose with chitosan and we are postulating that this peculiar shape will enhance the uptake of nanoparticles by macrophages.
Dialysis sac method was used to determine their lease of drug from nanoparticulate formulation.        The drug release kinetics was computed and it was observed to be following zero order kinetics (Table   10)

Conclusion
A promising targeted delivery system consisting of ligand conjugated Rifampicin nanoparticles incorporated in in situ gel was developed to tackle the drawbacks associated with the conventional treatment of osteoarticular tuberculosis. Rifampicin loaded mannose conjugated chitosan nanoparticles were prepared by ionic gelation technique. Mannose conjugated Chitosan nanoparticles showed preferential drug release in endosomal fluid indicative of preferential uptake of nanoparticles by macrophages and drug release in its endosomal contents. In vitro drug release of mannose conjugated Rifampicin nanoparticles incorporated in in situ gel system showed prolonged release for 40 hours. Based on the initial encouraging results obtained, the nanoparticles are being envisaged for ex-vivo cellular uptake study using TB infected macrophages.
ETHICAL STATEMENT: § Ethics approval and consent to participate: NA § Consent for publication: NA § Availability of data and materials: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. § Competing interests: The authors have no conflicts of interest/ competing interest to declare that are relevant to the content of this article. § Funding: The authors did not receive support from any organization for the submitted work. § Authors' contributions: All authors contributed to the study and manuscript conception and design.
• Material preparation, data collection and analysis were performed by Ms Pratiksha Prabhu.

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The research activity planning, execution and supervision was carried out under the guidance of Dr Sujata Sawarkar and Dr Pramila Chaubey. The first draft of the manuscript was written by Dr Sujata Sawarkar. Revisions and editing was done by Ms Trinette Fernandes. • All authors commented on previous versions of the manuscript, read and approved the final manuscript. § Acknowledgements: The authors wish to thank Sophisticated Analytical Instrument Facility, IIT-Bombay, Powai for their providing SEM analysis samples which are used in this manuscript.