Introduction

Tuberculosis (TB), caused by the bacterial species Mycobacterium tuberculosis, is endemic in India and several other developing countries1. Although it most commonly affects the lungs, TB can also be present in different forms, such as lymphadenitis, leptomeninges, radiculomyelitis, and spinal or renal tuberculosis2. Often referred to as a “medical chameleon” due to its varied clinical presentations, TB remains one of the few treatable diseases with an unacceptably high mortality rate3. The COVID-19 pandemic has unfortunately reversed the years of progress in reducing TB mortality, jeopardizing the gains made until 20194. The WHO Worldwide TB Report 2022 estimates that 10.6 million new cases of TB occurred in 2021, making it a continuing global health concern5. This setback is expected to increase TB fatalities in the next coming years, as prolonged, untreated cases may lead to further transmission and a significant rise in future TB cases6.

Treatment for this severe infectious disease typically involves a combination of drugs: Rifampicin, Isoniazid, Pyrazinamide, and Ethambutol, commonly referred to as the standard RIPE regimen. Among these, Rifampicin (RIF) is particularly significant due to its sterilizing capability, which helps to prevent relapses7. Its therapeutic efficacy in drug-susceptible TB is achieved by inhibiting bacterial RNA synthesis, acting as a transcriptional inhibitor by sterically blocking the elongation of the RNA transcript at the 5’ end8. The standard dosage of Rifampicin is usually 600 mg daily, for maintaining serum concentrations above the minimum inhibitory concentration (MIC) for M. tuberculosis. However, this dosage may be sub-optimal, as higher doses of approximately 20 mg/kg have been well tolerated and demonstrated more potent bactericidal activity than the standard 600 mg dosage9. Concerns about increasing the dosage often stem from the risk of serious side effects, such as thrombocytopenia, which has been linked to intermittent high-dose Rifampicin administration10. Nevertheless, addressing the need to shorten the Rifampicin administration period is crucial, given the growing issue of drug resistance. Another key antibiotic in treating tuberculosis (TB), is Isoniazid (INH), which is well-known for its bactericidal action against M. tuberculosis. As a prodrug, it inhibits mycolic acid production, an essential component of the mycobacterial cell wall, when activated by the bacterial enzyme KatG. Despite its effectiveness, Isoniazid-resistant TB strains present serious obstacles to international TB control initiatives11. The goal of several ongoing research works is to enhance Isoniazid’s therapeutic efficacy and address the resistance issues. To improve treatment outcomes, experiments are being conducted on the pharmacokinetics and pharmacodynamics of high-dose Isoniazid against the Isoniazid-monoresistant TB12.

In biomedical pharmaceuticals, an in-situ gel system (IGS) is a formulation that experiences a phase transition, changing from a liquid to a gel state upon application within the body, in response to environmental factors such as temperature, pH, or concentration. The scope of in-situ gels lies in their ability to provide sustained drug delivery, enhance bioavailability, and improve therapeutic outcomes by addressing various challenges, particularly through mucosal, ophthalmic, topical and parenteral (intramuscular and subcutaneous) routes of application13,14.

Poloxamer, well-known for its thermo-sensitive gelation properties, facilitates a streamlined injection process, leading to gel formation at the injection site15. Poloxamer 407’s biocompatibility, coupled with its successful use in pharmaceutical applications, underlines its relevance for intramuscular delivery of the drugs16. Additionally, Carbopol is recognized for its pH-sensitive hydrogel-forming capabilities, which play a crucial role in gel viscosity and gel strength. Its ability to quickly form a gel matrix upon injection facilitates a more sustained release of the drugs, ensuring stable drug levels for the required period17. Moreover, the biocompatibility and stability of Carbopol 934 enhance its suitability for incorporation into pharmaceutical formulations intended for chronic treatment18. These key features of the polymers support extended therapeutic effects, potentially reducing dosing frequency and thereby improving patient compliance with the treatment regimen19. Concurrently, the study also explores the potential of Hydroxy Propyl Methyl Cellulose (HPMC) as a biocompatible hydrophilic polymer with its gel-forming properties, allowing for greater control of drug release within muscle tissue, thereby maintaining therapeutic concentrations over a required duration of time. The stability of HPMC, combined with its tunable release kinetics and favorable safety profile, makes it a strategic choice for optimizing the dosage form20.

An in-situ gel-based intramuscular sustained release formulation combines the benefits of a gel texture with slow release mechanisms by delivering the drugs directly into the muscle tissue. This approach aims to provide prolonged release of the active ingredients, potentially leading to more consistent therapeutic concentration of the drug and reducing the frequency of administration compared to regular injections, which can improve patient comfort, compliance, and overall treatment adherence. The gel matrix, composed of polymers or other gel-forming materials, encapsulates and retains the active ingredients, facilitating their gradual release over an extended period. Optimizing the formulation is essential to ensure gel consistency, drug solubility, and compatibility with the injection procedure apparatus, such as syringes. Additionally, production practices and stability factors must be carefully considered to maintain the integrity of the gel medium and the drug’s efficacy7,21. Henceforth, in-situ gel-based slow-release formulations are designed to achieve specific release profiles to maintain steady drug levels in the bloodstream based on the drug’s therapeutic requirements, allowing for customization to match the desired dosing interval and pharmacological effects, leading to better treatment outcomes and potentially minimizing the side effects associated with rapid fluctuations in drug levels22.

With this background, the primary objective of this study is to develop and optimize an in-situ gel formulation with Poloxamer 407 serving as the primary polymer that can sustain the release of anti-tubercular drugs Rifampicin and Isoniazid, for an extended period. The study includes the selection of suitable polymers using molecular docking, optimization of gelation properties, physicochemical characterization of dual drugs loaded formulations, in-vitro drug release studies and their kinetics. The novelty of the work lies in the optimization of the in-situ gelling polymers using molecular docking method with the combination of dual drugs to formulate injectable sustained release system. This optimized product is expected to offer sustained release of the drugs, suitable for parenteral administration, high stability and non-toxic in nature.

Materials and methods

Materials

The Poloxamer 407 (Kolliphor®) was purchased from Sigma Aldrich in Mumbai, India. HPMC with a molecular weight of 86 kDa, Carbopol 940, Dialysis membrane-110, and pure Rifampicin were obtained from HiMedia Laboratories Pvt. Ltd in Mumbai, India. Isoniazid was sourced from Tokyo Chemical Industries in Japan. All other materials used were of analytical grade.

Methods

Molecular docking studies

Ligand preparation

The structure of Isoniazid (PubChem ID: 3767) was downloaded from PubChem. This compound was then modified by adding specific functional groups using the 2D Sketcher module in the Schrodinger Materials Science Suite (v.2024-4)23. Then, the compound structure was subsequently prepared using the LigPrep module24. The preparation process included generating possible ionization states at a target pH of 7.0 ± 2.0 using the ionizer, tautomerization, desalting, and optimization. Structural minimization was conducted using the OPLS4 force field25.

Polymer preparation

Monomeric compound coordinates for the polymers were obtained for HPMC (PubChem ID: 57503849)26, poloxamer 407 (PubChem ID: 10154203), and acrylic acid, which was directly modeled using the Polymer Builder suite. All downloaded monomeric structure coordinates were converted to 3D using the LigPrep module and minimized with the OPLS4 force field in the Schrodinger suite. The polymer structures were then constructed using the Polymer Builder module in the Schrodinger Materials Science Suite. The monomers were added in different compositions, as specified in (Supplementary Table S1). An amorphous cell was generated for each polymer using the OPLS4 force field, resulting in a total of 15 distinct polymers being constructed.

Binding site prediction

The Site Map module27 was used to analyze the polymer structures and predict binding sites with a high probability of ligand interaction. Potential binding sites were identified and characterized based on their size, shape, hydrophobicity, and capacity for hydrogen bonding. Binding sites corresponding to 15 polymers were generated and these sites were ranked according to their SiteScore, wherein the highest-ranked binding sites were selected for subsequent docking studies.

Grid generation

Receptor grids were created using the Receptor Grid module in Glide28 for the docking studies. The highest-scoring binding sites identified by Site Map for each polymer was chosen as the center of the grid. The grid box was positioned around the center of the selected ligands to ensure that the entire binding site was included. A total of 13 grids were generated corresponding to each polymer.

Molecular docking

Molecular docking was performed using the prepared ligand structure and the receptor grids generated for each polymer composition. The docking process was conducted with the Glide Dock module28 in extra precision (XP) mode to ensure high accuracy in ligand-receptor interactions. During the docking simulations, the ligands were positioned in the predicted binding sites within each polymer, and their binding affinities were calculated. The docking results offered insights into the binding modes, interaction energies, and stability of the ligand-polymer complexes.

Optimization of in-situ gel formulations

The in-situ gel (ISG) formulations were prepared by the cold method29,30, by combining Poloxamer 407, Carbopol 940, and HPMC. Based on the docking score, Poloxamer 407 was fixed at a concentration of 20% w/v, serving as the primary gel base polymer. The concentrations of HPMC and Carbopol 940 were varied between 0.5–2.0% w/v and 0.5–0.75% w/v, respectively (concentration fixed from the docking score—Supplementary Table S1), to achieve a product that exhibits optimum gelling at body temperature. Through sol–gel transition studies, the appropriate levels of HPMC and Carbopol 940 were identified and optimized. The compositions of all the in-situ gel trial formulations are provided in Supplementary Table S2. Briefly, Solution 1 contains Carbopol dissolved in sterile double-distilled water at room temperature, and Solution 2 contains Poloxamer 407 and HPMC dissolved under ice-cold conditions. The polymers were added based on the percentage weight by volume basis to obtain the specific concentrations. Both solutions were gradually mixed and bath sonicated to obtain a homogeneous, clear mixture. Based on observations and optimizations, the ISG1 and ISG5 formulations were identified with optimum parameters, and so, considered suitable for drug loading and further evaluation studies. The ISG1 formulation consisted of 20%w/v Poloxamer 407, 0.5%w/v HPMC and 0.5%w/v Carbopol 940, while the ISG5 formulation contained 22%w/v Poloxamer 407, 0.4%w/v HPMC and 0.6%w/v Carbopol 940. The anti-tubercular drugs Rifampicin and Isoniazid were added individually or in combination, at concentrations of 10 mg/mL and 5 mg/mL (based on therapeutic dosing as 2:1), respectively. The final products were stored at 4 ºC for 24 h before undergoing evaluation studies.

Determination of gelation temperature

The gelation temperature is the condition at which the sol gets converted into a non-flowable gel form, which was determined by the tube inversion method. The gelation temperature of the formulated in-situ gels was evaluated using the test tube inversion method. The refrigerated formulation sample of 2 mL (in solution state) was taken in a 15 mL test tube and placed in a water bath (Labline Ultra Cryostat Bath, Labline Instruments, Cochin, India). The temperature was gradually raised by 1 °C/min, and gelation was observed at above 20 °C. The lowest temperature at which the meniscus did not move upon tilting the test tube to a 90° angle for 30 s was recorded as the gelation temperature31. The experiment was performed in triplicate for all samples, and the average with standard deviation (SD) was calculated.

Gel erosion study

The gel erosion study was conducted by the gravimetric method, where the gel matrix could be degraded/disintegrated under physiological conditions, releasing the monomer or drug. It is important to establish the mechanical strength of the in-situ gel formulations32. The stability of the formulations was observed at physiological pH and body temperature. About 5 g sample was placed in a 20 mL amber glass vial, weighed, and stored at 37 °C. Then, 1 mL aliquot of 0.133 M Sorensen’s phosphate buffer (pH 7.2) was added to the sample. At 30-min intervals, the exposed buffer was separated and replaced with fresh buffer in the gel sample. After removing the buffer, the weight of the remaining gel was measured to assess the mass loss due to erosion33. This process was continued for up to 7 h, and the data were collected in triplicate to record the mean with standard deviation.

Drug content

The homogeneity of drug concentration among all formulations (ISGRIF2, ISGINZ3, ISGRIFINZ4, ISGRIF6, ISGINZ7 and ISGRIFINZ8) was assessed by dissolving a standard volume of each formulation in phosphate buffer, followed by appropriate dilution, and detecting the drugs using UV–visible spectrophotometry at 334 nm for Rifampicin and 261 nm for Isoniazid34. Accurately, 1 mL of the sample formulation was placed in a 100 mL volumetric flask, mixed thoroughly with 50 mL of phosphate buffer (pH 7.4), sonicated, vortexed, and finally brought to the volume with the buffer. The solution was incubated overnight and then filtered. The absorbance of the samples was quantified using a UV spectrometer (UV 1800, Shimadzu Corp, Japan) against the respective buffer blank, and the drug content was calculated based on the standard calibration curve plot of the pure drugs35.

Viscosity of the in-situ gel at the sol state

The viscosity of the in-situ gel formulations at the sol condition was measured using a viscometer (Brookfield DV-II + Pro EXTRA, Brookfield, USA) with spindle 63 at various shear rates, including 30, 50, 60, 100, 150, and 200 rpm34. The spindle was inserted perpendicularly into the sample and rotated at a constant angular speed for 30 to 60 s at each shear rate. The viscosity of all formulations was measured while maintaining the sample temperature at 4 °C. The procedure was performed in triplicate to obtain the mean with standard deviation (SD).

Viscosity of the in-situ gel formulations at the gel state

The rheological properties of the gel samples were characterized using an Automatic Rheometer (Anton Paar MCR 302, USA). The viscosity and flow behavior of all formulations were measured at a shear rate of 50 across a temperature range of 25 °C to 37 °C. The sample was placed on the lower plate with a diameter of 40 mm and was allowed to equilibrate for one minute. The probe was positioned perpendicularly in contact with the sample. The oscillatory parameters were set to observe the readings to understand the rheological behavior36.

Fourier transform infrared spectroscopy analysis

Fourier Transform Infrared Spectroscopy (FT-IR) (Perkin Elmer Spectrum 100, USA) was used to investigate potential interactions between the drugs and polymers37. The compatibility and integrity of the formulations were assessed by analyzing the infrared spectrum of freeze-dried formulations compared to the pure drugs and polymers (Poloxamer 407, HPMC, and Carbopol 940) using the KBr technique. The samples were mixed with anhydrous potassium bromide and formed into pellets using a hydraulic press at a pressure of 53.62 kp N cm−2, and analyzed in the transmission mode between 4000 and 400 cm−1 wavenumber.

Morphology characterization using scanning electron microscope (SEM)

Scanning Electron Microscopy (SEM) is a high-resolution imaging technology used to study the surface morphology of materials. SEM (Vega3, TESCAN, Czech Republic) was utilized to observe the freeze-dried samples of in-situ gel formulations, compared to the individual pure components. The samples were mounted on an aluminum stub using double-sided carbon tape, and a sterile spatula was employed to position the sample on the tape. The stub setup was sputter-coated with a thin gold film to ensure conductivity for visualizing three-dimensional images of the samples38. Imaging was conducted at a voltage of 10 kV, capturing images at various magnifications ranging from 200 to 20,000X.

Thermal behavior of the in-situ gel formulations using TG-DSC

The thermal behavior of the polymers, anti-tubercular drugs (Rifampicin & Isoniazid), and in-situ gel formulations was characterized using thermogravimetry and differential scanning calorimetry (TG-DSC) (SDT Q600). Approximately 3 mg of each sample was placed in an aluminum pan, sealed, and heated from 0 to 600 °C at a heating rate of 10 °C/min. TG-DSC thermograms were compared to identify significant changes due to the interaction of drugs with polymers in the formulations.

In-vitro drug release study

The in-vitro drug release studies were conducted using a dialysis membrane-110 (cut off: 12–14 kDa) soaked in phosphate buffer (pH 7.4). A 30 mL vial was filled with 10 mL of phosphate buffer (pH 7.4). Precisely 1 mL of the in-situ gel formulation (containing RIF: 10 mg/mL and INZ: 5 mg/mL) was placed in a dialysis bag and secured at both ends. The sample-containing bag was immersed in the buffer. The entire setup was assembled on a thermostatic magnetic stirrer and stirred continuously at 100 rpm, maintained at 37 °C for 10 days. The drug-released samples were collected every hour and replaced with 10 mL of fresh buffer to uphold the seamless sink conditions. The collected samples were measured at 334 nm for Rifampicin and 261 nm for Isoniazid using a UV–visible spectrophotometer (Shimadzu UV 1800, USA)34. All sample experiments were conducted in triplicate and compared to the in-vitro release profile of unprocessed drug samples containing an equivalent drug concentration33,39.

In-vitro cell toxicity assay

The in-vitro cytotoxicity assay of the in-situ gel formulations was assessed using fibroblast cell lines L929 by the MTT assay, based on a standardized protocol with minor modifications37. The monolayer cells were detached using trypsin-ethylenediaminetetraacetic acid (EDTA) to prepare a single-cell suspension. The viable cells were counted with a hemocytometer, diluted with medium containing 10% fetal bovine serum (FBS), and adjusted to a final 1 × 105 cells/mL concentration. About 100 µL of this cell suspension was plated into each well of a 96-well plate, achieving a density of 10,000 cells per well. The plates were incubated at 37 ºC, with 5% CO2, 95% air, and 100% relative humidity to allow cell attachment. After 24 h, the cells were exposed to serial concentrations of test samples ranging from 6.5 to 100 µg/mL. The plates were then incubated for 48 h under the same conditions. Following this, 15 µL of MTT in phosphate-buffered saline (5 mg/mL) was added to each well, and the plates were incubated at 37 °C for another 4 h. After incubation, the MTT-containing medium was removed, and the formazan crystals were dissolved in 100 µL of DMSO. The absorbance was then measured at 595 nm using a microplate reader37.

Ex-vivo permeation study

For the ex-vivo permeation study, a Franz diffusion cell was utilized, with the receptor compartment filled to the brim with phosphate buffer (pH 7.4). The donor chamber was positioned above the receptor chamber, and a cleaned buccal membrane from a freshly slaughtered goat was placed on top40. The setup was stabilized at 37 °C and maintained at 100 rpm. About one mL of the formulation sample (containing RIF: 10 mg/mL and INZ: 5 mg/mL) was placed in the donor chamber over the membrane, and samples were collected from the receptor chamber through the side port at time intervals of 0.5, 1, 1.5, 2, 3, 4, 5, and 6 h. After collecting approximately 2 mL of sample at each time point, the receptor chamber was replaced with fresh phosphate buffer (pH 7.4) to maintain sink conditions. The collected samples were measured using a UV–visible spectrometer (Shimadzu UV 1800, USA) at 335 nm and 261 nm for Rifampicin and Isoniazid, respectively38. The cumulative amount of drug permeated across the biological membrane and flux was estimated from the data and compared to the pure drugs considered as a reference control40.

Drug release kinetics

By fitting the drug release data obtained for phosphate buffer media to various release kinetic simulation models, including zero order, first order, Higuchi, Hixson–Crowell, Korsmeyer–Peppas model, Makoid-Banakar, Gompertz, Weibull, Hopfenberg, and Baker Lonsdale, the drug release mechanism of the in-situ gel with anti-tubercular drugs was investigated. The best-fitting model for each trial is the one with the lowest sum of squared residuals (SSR) and the highest correlation coefficient (R2 value). A plug-in program called DD Solver was used to simulate the drug release41,42.

Statistical analysis

All the experiments were carried out in triplicate. The outcome results were analyzed using GraphPad Prism 6.0 software and Origin 2018 and presented as mean ± standard deviation. Data from the cell viability assay was analyzed using an unpaired Student’s t-test.

Results and discussion

Optimization of in-situ gel formulation

As the release of the drug was anticipated for several days, the combination of Carbopol and HPMC with Poloxamer was chosen to enhance the encapsulation of the drugs. We have utilized a molecular docking study to assess the affinity of the drugs to polymers of different compositions. A higher negative score implied a strong affinity, and hence the polymer could retard the drug slowly, enabling a sustained release pattern. The percentage of polymers with Isoniazid was determined based on the docking score obtained from the Schrodinger suite of programs and was used to initiate the formulation preparation. Since the cyclic structure of Rifampicin was not acceptable for docking, Isoniazid was selected, and a systematic study was commenced by varying the concentrations of the components. Initially, Poloxamer was selected based on its sol-to-gel behavior, and its concentration was varied from 18%w/v to 25%w/v using water as the medium. Based on the docking scores (− 3.432 to − 3.085 kcal/mol), the optimal concentration of poloxamer was found to be 22%w/v. After fixing the concentration of poloxamer at 22%w/v, the concentration of Carbopol was varied from 0.5%w/v to 2.5%w/v. A similar approach was applied to HPMC, which also varied within the range of 0.5%w/v to 2.5%w/v. The docking score improved with an increase in Poloxamer concentration up to 25%w/v. Although the docking score for 24%w/v was 4.629, the gelation temperature was below 25 °C32. Since the docking scores for 20%w/v and 22%w/v of Poloxamer are − 3.432 and − 3.085, respectively, the 22%w/v was selected, and the binding module with varied percentages of carbopol was assessed. The compositions that included carbopol from 0.2%w/v to 0.6%w/v showed similar docking scores. However, beyond 0.6%w/v of carbopol, no favorable pose was found (Table 1), suggesting poor binding. In a similar manner, HPMC also demonstrated binding scores at its optimum concentration range. Consequently, the preparation of formulations commenced with carbopol at 0.6%w/v or below, and HPMC at 1–2%w/v, based on sol-to-gel temperature observations and docking scores with the primary polymer, poloxamer. The formulations were trialed by varying the composition of carbopol and HPLC with fixed Poloxamer concentrations at 20%w/v and 22%w/v to assess the sol–gel transition effects. Experimental optimization of in-situ gel formulations involved trials of various Poloxamer 407, HPMC, and Carbopol 940 combinations, as detailed in (Supplementary Table S2). These formulations were further loaded with the antitubercular drugs Rifampicin and Isoniazid. Poloxamer 407 was chosen for its in-situ gel formation capability, essential for the sustained release of the drugs35, and its combination with either Carbopol or HPMC has been explored to tailor the rheology of the product and to improve the stability of the formulations29,43.

Table 1 Optimization and physicochemical characterization of anti-tuberculosis drugs loaded in-situ gel formulation.
Full size table

Gelation temperature

Gel formation under in vivo conditions (37 ± 0.5 °C) is considered as most suitable for achieving sustained drug release. The concentration of Poloxamer 407 and the ratio of Carbopol 940 to HPMC influenced the gelation temperature. An increase of HPMC between 1–2%w/v in the Poloxamer (20–22%w/v) gels resulted in a 1 °C decrease in the sol–gel temperature for every 0.5%w/v change. Based on the understanding from in-silico work and trial formulations, the Carbopol concentration was verified at 0.75%w/v, 0.6%w/v, and 0.5%w/v, with HPMC being less than 0.5%w/v and Poloxamer maintained at 20–22%w/v.

The trial formulations 8, 9, 11, and 12 (Supplementary Table S2) were selected for further studies due to the observed gelation temperature. Formulations 8 and 9 (Supplementary Table S2), which contain 20%w/v poloxamer and 1%w/v of Carbopol and HPMC blend, had gelation temperatures of 30 °C and 31 °C, respectively. However, with the identical amounts of Carbopol and HPMC, an increase of 2%w/v poloxamer resulted in decreased gelation temperatures of 26 °C and 28 °C. Also, the addition of the drugs did not alter the sol-to-gel transition temperature.

Gel erosion study

The erosion study aimed to understand the degradation of the gel over time, influencing drug release patterns. The formulation ISG1, which contains PLX 20%w/v with Carbopol and HPMC in a 50:50 ratio, exhibited a higher gel erosion rate of 47.5% compared to formulation ISG5, which has PLX 22%w/v with Carbopol and HPMC in a 60:40 ratio, showing a lesser erosion of 33.6% over 7 h. The greater erosion observed in ISG1 formulation may be attributed to a lower percentage of Poloxamer and equal ratios of Carbopol and HPMC, while ISG2 contains a higher amount of the gelling polymer Poloxamer, in addition to a greater quantity of the hydrophobic polymer Carbopol and a lower amount of the hydrophilic polymer HPMC. The formulations loaded with individual drugs (ISGRIF2 and ISGINZ3) or a combination of drugs (ISGRIFINZ4 and ISGRIFINZ8) exhibited a minor difference in the rate of erosion32. The percentage of erosion of the in-situ gels was observed to be in the following order:

ISG1 > ISGRIFINZ4 > ISGINZ3 > ISGRIF2 > ISG5 > ISGINZ7 > ISGRIFINZ8 > ISGRIF6 (Fig. 1).

Fig. 1
Fig. 1
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Gel erosion study of two different sets of in-situ gel formulations (n = 3) (a) In-situ gel formulations Blue circle: ISG1, Blue square: ISGRIF2, Blue triangle: ISGINZ3, and Red inverted triangle: ISGRIFINZ4 (b) In-situ Formulations Blue circle: ISG5, Blue square: ISGRIF6, Blue triangle: ISGINZ7, and Red inverted triangle: ISGRIFINZ8.

Drug content

The presence of both drugs in a homogeneous distribution, without significant chemical modifications and high stability, is one of the crucial parameters for the effectiveness of the formulation. The ISG1 formulation loaded with individual drugs showed an average drug content of 104 ± 2.80% for ISGRIF2 and 101 ± 1.07% for ISGINZ3. When the individual drugs were added to the ISG5 formulation, the drug content was quantified to be 100 ± 2.17% and 97 ± 1.31% for ISGRIF6 and ISGINZ7, respectively. The formulations loaded with both the drugs exhibited drug content of 100 ± 4.97%, 99 ± 1.92%, 103 ± 2.70%, and 95 ± 2.32% for ISGRIFINZ4 (Rifampicin), ISGRIFINZ4 (Isoniazid), ISGRIFINZ8 (Rifampicin), and ISGRIFINZ8 (Isoniazid), respectively. The content of both Rifampicin and Isoniazid calculated in the range of 95–105%, confirmed the stability of the drugs in the selected formulations.

Viscosity analysis

Viscosity analysis of the formulations has been conducted to confirm that the sol-to-gel transition could correspond with quantifiable changes in fluidity and viscosity. The sol viscosity of the formulations IG01 and IG02 ranged between 79–155 cP and 87–238 cP at 4–5 °C, respectively, while the gel viscosity was measured to be 870–1499 cP for ISG1 and 1000–1700 cP for ISG5 at 37 °C. The in-situ gel formulations exhibited pseudoplastic behavior, showing that an increase in shear rate from 30–200 rpm had a significant decrease in viscosity (Supplementary Fig. S1). The sol viscosity was found to be suitable for loading the formulation into a syringe and facilitating administration as an intramuscular injection, followed by gel formation in-situ, wherein the gel viscosity was appropriate for providing sustained drug release. The sol viscosity of the formulations loaded with individual drugs and combinations of drugs ranged from 80 to 200 cP, indicating no statistically significant difference compared to the plain in-situ gels (without drugs). Similar results had been previously reported, where the sol viscosity of 15–20%w/v poloxamer 407-based formulations ranged from 18 to 28 cP, and the gel viscosity was 1046–6179 cP at 200–10 rpm2,34. Likewise, another report stated about a periodontal in-situ gel formulation with 15–20%w/v poloxamer 407 and > 0.5%w/v carbopol 934P, showing a gel viscosity up to 30,596 cP at body temperature35. An analogous study with Secnidazole in-situ gel formulation prepared using Carbopol 940 and HPMC K4M at 0.45%w/v each, had demonstrated the sol viscosity of 24 cP, comparable to the concentration of the polymers used in our ISG5 formulation37.

Rheology

Rheological analysis of the in-situ gels had confirmed the non-Newtonian flow behavior and shear thinning property, supporting their suitability for intramuscular administration and stability for sustained drug release44,45 (Fig. 2). In case of the in-situ gel formulations prepared with 20%w/v Poloxamer 407, 0.5%w/v Carbopol 940, and 0.5%w/v HPMC, there was no significant difference observed between ISGRIF2 and ISGINZ3 formulations, indicating the flow behavior was independent of the separately loaded drugs. However, the formulations ISGRIFINZ4 and ISGRIFINZ8 had shown higher viscosity due to the presence of two drugs and their interaction with the three polymers, wherein the values were variable with high error bars. In case of the formulations developed with 22%w/v Poloxamer 407, 0.6%w/v Carbopol 940, and 0.4%w/v HPMC, the resistance to flow was comparatively greater due to a higher amount of hydrophobic polymers, which is one of the primary contributing factors for the gelling36,46.

Fig. 2
Fig. 2
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Viscosity of in-situ gels loaded with individual drugs and combination (a) Formulations containing 20%w/v Poloxamer 407, 0.5%w/v Carbopol 940 and 0.5%w/v HPMC; (b) Formulations containing 22%w/v Poloxamer 407, 0.6%w/v Carbopol 940 and 0.4%w/v HPMC.

FTIR analysis

FTIR characterization of the in-situ gel formulations loaded with the drugs was compared to the pure drugs Rifampicin and Isoniazid, as well as the pure polymers Poloxamer 407, Carbopol 940, and HPMC, for identification of the functional groups present in each sample. The FT-IR spectrum of the native drug revealed distinctive peaks at characteristic wave numbers, identifying the molecule’s functional groups at 3480 cm−1 for O–H stretching of alcohol, 2939 cm−1 for C–H stretching of alkane, 2826 cm−1 for C–H stretching of aldehyde, 1643 cm−1 for C=C stretching of alkene, and 1217 cm−1 for C–O stretching. The characteristic peaks obtained for the polymers at 3418 cm−1, 2885 cm−1, 2238 cm−1, and 1110 cm−1 correspond to intermolecular O–H stretching of alcohol, C–H stretching of alkane, C≡C stretching, and C–O stretching, respectively (Fig. 3). The freeze-dried sample of in-situ gel containing the drug showed typical polymer peaks with slight shifts in the drug peaks, indicating minimal drug-polymer interactions. The characteristic peaks observed in the native drug and polymers in the range of 3000–4000 cm−1 were slightly shifted with a reduction in intensity, indicating that the polymers underwent intermolecular hydrogen bonding with the drugs. The IR spectrum of Carbopol is characterized by an absorption peak at 3089 cm−1 (O–H stretching) and 1706 cm−1 (carboxyl group). The poloxamer FTIR spectrum showed principal absorption peaks at 2893 cm−1 (C–H stretch aliphatic), 1355.86 cm−1 (in-plane O–H bend), and 1124.42 cm−1 (C–O stretch). The IR spectrum of Rifampicin indicated absorption peaks at 1728 cm−1 (Carbonyl group), 1650 cm−1(–C = N– asymmetric stretching group), and 1566 cm−1 (Amide group)47. The FTIR spectra of the in-situ gel formulations showed minimal shifts in drug peaks, indicating weak drug-polymer interactions. This confirmed the encapsulation of drugs within the polymer matrix without significant chemical alterations.

Fig. 3
Fig. 3
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FTIR Spectrum of in-situ gel formulations compared to native drugs and pure polymers (a) Rifampicin loaded in-situ gels (ISGRIF2 & ISGRIF6), (b) Isoniazid loaded in-situ gels (ISGINZ3 & ISGINZ7), and (c) Dual drug loaded in-situ gels (ISGRIFINZ4 & ISGRIFINZ8).

SEM analysis

Scanning Electron Microscopy (SEM) revealed the non-uniform powder structure of the native drugs, Rifampicin and Isoniazid (Fig. 4). The ISG1 and ISG5 formulations loaded with Rifampicin and Isoniazid, exhibited modifications in the morphological characteristics of the drugs, evidenced by the disappearance of specific powder particles, due to their encapsulation within the gelling polymer matrix, which led to the masking of the original physical properties of the drugs. In case of the in-situ gel formulations, an orderly pattern of entangled polymer gel matrix could be observed, which may be because of the freeze drying of hard-sphere crystal lattice unique structure of triblock copolymer formed by the aggregation of Poloxamer micelles based gels48.

Fig. 4
Fig. 4
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Surface morphology images of native drugs and the in-situ formulations incorporated with the drugs (a) Rifampicin, (b) Isoniazid, (c) ISGRIFF2, (d) ISGINZF3, (e) ISGRIFINZF4, (f) ISGRIFF6, (g) ISGINZF7, and (h) ISGRIFINZF8.

TG-DSC analysis

Thermogravimetric analysis (TG) and Differential Scanning Calorimetry (DSC) revealed the thermal behavior changes of the in-situ gel formulations with respect to heat flow and weight changes, as compared to the pure drugs and polymers. The DSC thermogram of the native Rifampicin sample showed a characteristic exothermic peak at 260 °C, followed by an endothermic melting peak at 305 °C indicating the thermal decomposition by melting (Fig. 5a). Correspondingly, an initial sudden weight loss was observed, followed by a gradual decrease of the mass in the TG curve49. In the case of a pure sample of Isoniazid, the DSC thermogram displayed the characteristic sharp melting point at 171 °C, and a corresponding significant weight loss in the TG curve, indicating the typical melting point of the drug (Fig. 5b)50,51. The polymer PLX 407 sample exhibited a clear pattern of melting temperature between 57 and 60 °C (Fig. 5c).

Fig. 5
Fig. 5
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Thermogravimetric and differential scanning calorimetry (TG-DSC) curve of the in-situ gel formulations compared to pure drugs and polymer (a) Rifampicin, (b) Isoniazid, (c) Poloxamer, (d) ISGRIF2, (e) ISGINZF3, (f) ISGRIFINZ4, (g) ISGRIF6, (h) ISGINZF7, and (i) ISGRIFINZ8.

The DSC thermogram of lyophilized in-situ gel samples containing poloxamer 407 and Rifampicin showed a critical endothermic peak at 59 °C for the melting of the polymer, followed by a non-linear degradation curve from 300 to 426 °C (Fig. 5g), wherein the drug peak was shifted from its original melting point range. The TG curve of the in-situ gel demonstrated a corresponding gradual, stepwise weight loss up to 426 °C. These changes are attributable to its encapsulation within the polymer matrix, which has enhanced the thermal stability of the drug in the formulation, with slower degradation than pure Rifampicin. A similar phenomenon was observed in the TG-DSC pattern of Isoniazid loaded in-situ gels. In case of the in-situ gels loaded with both the drugs, the characteristic peaks of both Rifampicin and Isoniazid were slightly shifted from their original values, wherein the sharp endothermic peaks were modified as broader and less intense peaks. The decomposition temperature increased for drugs due to a delay in the onset of melting, indicating the successful incorporation of both drugs within the polymer matrix, which likely provided a protective effect. Also, this could be because of the decrease in the crystallinity of the drugs loaded in the polymer blend matrix and the mild interactions between the two drug molecules, and also within the drugs-polymers52,53. These results provide evidence for effective encapsulation of drugs and the formation of a stable drug-polymer complex within the in-situ gel system.

In-vitro drug release study

The study aimed to develop a model gel capable of releasing the drugs for up to a week, based on the interaction between three polymers. The in-vitro release studies conducted over 10 days demonstrated the sustained release capabilities of the ISG1 (containing 20%w/v poloxamer 407, 0.5%w/v Carbopol 940 and 0.5%w/v HPMC) and ISG5 (containing 22%w/v poloxamer 407, 0.6%w/v Carbopol 940 and 0.4%w/v HPMC) formulations. The ISG1 formulation with a lower concentration of Poloxamer 407 showed a faster drug release, particularly at pH 7.4, whereas ISG5 with a higher concentration of Poloxamer 407, exhibited a prolonged release due to improved drug entanglement within a greater amount of polymer matrix and formation of a stronger, stiffer in-situ gel with reduced erosion under physiological pH conditions. Comparison of the two drugs Rifampicin and Isoniazid release profiles had clearly demonstrated a distinct pattern, due to their varying level of solubility and dissolution54.

When Rifampicin was loaded in the in-situ gel individually, the drug was released within 32 h from the formulation ISGRIF2, however extended further in formulation ISGRIF6 until 226 h (approximately 9 days) (Fig. 6a). This could be attributed to the slow linear release of the hydrophobic drug from the triple blend polymer matrix gel containing higher percentage for poloxamer and Carbopol55,56. In case of Isoniazid, there was a rapid release within 1 h from the formulation ISGINZ3, which was almost similar to the pure drug, which could be due to the highly hydrophilic nature of the drug, which was not completely entrapped in the gel matrix containing a higher amount of HPMC polymer. However, there was a slower release of the drug in the formulation ISGINZ7 until 144 h, due to improved entanglement of the drug in the more hydrophobic polymer composition blend. Still, a burst release of about 70% drug was observed within the first hour (Fig. 6b), due to the high dissolution rate of the freely soluble drug57.

Fig. 6
Fig. 6
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In-vitro drug release study of native drugs individually, in combined form, and in-situ gel formulations filled with TB drugs (a) Pure Rifampicin, ISGRIF2, and ISGRIF6 (b) Pure Isoniazid, ISGINZ3, and ISGINZ7 (c) Pure Rif (Rif + Ind), ISGRIFINZ4 Rif (Rif + Ind), ISGRIFINZ8 Rif (Rif + Ind), Pure Ind (Rif + Ind), ISGRIFINZ4 Ind (Rif + Ind), and ISGRIFINZ8 Ind (Rif + Ind).

When both the drugs were loaded together in the in-situ gelling system, the dissolution of the drugs were mutually influenced by each other (Fig. 6c). In case of the formulation ISGRIFINZ4 (containing 20%w/v poloxamer 407, 0.5%w/v Carbopol 940, and 0.5%w/v HPMC), Rifampicin showed sustained release for about 128 h (5 days), whereas Isoniazid was released within a day, which was similar to the trend of pure drug combination. However, in the formulation ISGRIFINZ8 (containing 22%w/v poloxamer 407, 0.6%w/v Carbopol 940, and 0.4%w/v HPMC), Rifampicin showed more linear sustained release for about 288 h (equivalent to 12 days), and Isoniazid provided sustained for about 2 days. The release pattern of the combined drugs was different from the previously reported bilayer tablet formulation, wherein Rifampicin was released faster by immediate release layer and Isoniazid was retarded58.

Drug release kinetics

The in-vitro drug release kinetics were analyzed using DD-solver software to assess the appropriate model for explaining the mechanism of release of both drugs, Rifampicin and Isoniazid, based on the highest regression values and low residual error59. The kinetics study revealed that the best-fit model for the release of the two different drugs from the formulated in-situ gel was the Makoid-Banakar model, with the R2 value greater than 0.98 in all the trials. This demonstrated that the mechanism of drug release was chiefly influenced by the drug-polymer interactions. Additionally, the Weibull model, with a high R2 value (> 0.98), confirmed the drug release from the homogeneous matrix drug delivery system. The dual drugs loaded in situ gels had shown highest R2 value in the primary kinetic model, defined by Korsemeyer Peppas, wherein the release of Rifampicin was based on non-Fickian diffusion (n value > 0.5) and the release of Isoniazid followed Fickian (n value < 0.45) phenomenon60. In the case of pure Isoniazid loaded formulations, the Hixon-Crowell kinetics were identified with the highest R2 (0.99), indicating a sudden steep increase in the initial drug release, followed by a slow profile until reaching maximum release. This phenomenon could be due to the burst release of the hydrophilic drug from the freely eroding gel matrix containing less poloxamer and more HPMC (Supplementary Table S3)60,61.

Cell toxicity assay

The detrimental effect of in-situ gel formulation on the cell culture system was studied using the MTT assay with L929 cell lines. These cell lines are commonly used to assess the cytotoxicity of formulations. The formulation ISGRIFINZ8 contains both Rifampicin and Isoniazid at a 2:1 ratio. Cells treated with the freeze-dried sample of the in-situ gel formulation at concentrations ranging from 6.5 to 100 µg of Rifampicin and 3.12–50 µg of Isoniazid exhibited over 90% viability (Fig. 7). No significant toxicity was identified compared to the control untreated cell lines after 24 h. The study supported the use of the selected in-situ gel formulation for in-vivo experiments.

Fig. 7
Fig. 7
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MTT assay—Cell viability study using Fibroblast L929 cell lines (n = 3). (a) Rifampicin concentration (6.25–100 µg) (b) Isoniazid concentration (3.125–50 µg).

Ex-vivo permeation study

The ex-vivo permeation study conducted using a Franz diffusion cell for 6 h demonstrated that the pure individual drugs exhibited higher permeation compared to their combination, which may be due to the inhibitory effects of drug-drug interactions62,63. When the individual drugs were loaded in the in-situ gelling system ISGRIFF6 and ISGINZF7, there was significant improvement in the permeation of the drugs, which could be attributed to the amphiphilic nature of the polymer blend matrix carrier, which supported the entry of drug molecules across the biological membrane. In case of the formulation ISGRIFINZ8 loaded with two drugs, both the drugs exhibited higher permeation (50–60 µg/cm2) compared to the individual drugs loaded in-situ gels (ISGRIFF6 and ISGINZF7) showing lesser permeation (15–25 µg/cm2) until the end of 6 h (Fig. 8). This phenomenon evidenced a favorable performance due to the mutual drug-drug and drug-polymer interactions. The synergistic enhancement of permeation of both the drugs is advantageous to support the maximum uptake of the slowly released drugs in the biological environment64.

Fig. 8
Fig. 8
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Ex-vivo permeation study of Rifampicin and Isoniazid loaded in-situ gels compared to pure drugs (Blue circle: Rifampicin; Red square: Isoniazid; Blue triangle: ISGRIFF6; Inverted triangle: ISGINZF7; Pink circle: Rif (Rif + Inz) Control; unshaded circle: Inz (Rif + Inz) Control; unshaded square: Rif(IRFINZ8); Green triangle: ISGRIFINZ8.)

Conclusion

This study aimed to address the challenges in tuberculosis treatment by developing an in-situ gel for intramuscular administration, offering sustained drug release for up to 10 days. The formulations optimized with 22%w/v Poloxamer 407, 0.4%w/v HPMC, and 0.6%w/v Carbopol 940, and loaded Rifampicin and Isoniazid individually (ISGRIF6 & ISGINZ7) and together (ISGRIFINZ8), demonstrated promising results, particularly for the hydrophobic drug Rifampicin. Due to the hydrophilic nature of Isoniazid, the duration of release was shorter. However, there was a significant improvement in the permeation of both drugs. The in-situ gel demonstrated required sol viscosity to facilitate convenient injectability of the formulation, and sol to gel transition at optimum temperature under in vivo condition that favored the extended-release of the drugs. The product was optimized through molecular docking with specific combination of gelling agents. The ability of the injectable product to release the drugs for a more than a week, could reduce the need for daily administration, overcome the oral first pass hepatic metabolism, and enhance patient compliance, addressing the challenges associated with conventional dosage forms used for tuberculosis treatment. Henceforth, the developed injectable in-situ gel formulation could be recommended for parenteral administration to provide sustained release of Rifampicin and Isoniazid. Further, in vivo investigations are required to validate its therapeutic efficacy and safety, as well as exploring its scalability and potential applications in managing the multidrug-resistant tuberculosis.