Inhalable micro particle-based anti TB drug delivery systems are being investigated extensively for Tuberculosis [TB] treatment as they offer efficient and deep lung deposition with several advantages over conventional routes. It can reduce the drug dose, treatment duration and toxic effects and optimize the drug bioavailability. Yeast derived β-glucan is a β-[1–3/1–6] linked biocompatible polymer and used as carrier for various biomolecules. Due to presence of glucan chains, particulate glucans act as PAMP and thereby gets internalized by receptor mediated phagocytosis by phagocytes including macrophages. In this study, β-glucan microparticles were prepared by adding l-leucine as excipient, and exhibited 70% drug [Rifabutin] loading efficiency. Further, the sizing and SEM data of particles revealed a size of 2–4 µm with spherical dimensions. The FTIR and HPLC data confirmed the glucan composition of particles. The mass median aerodynamic diameter [MMAD] and Gravitational standard deviation [GSD] data indicated that these particles are inhalable in nature and have better thermal stability as per DSC thermogram. These particles were found to be non-toxic upto a concentration of 80µg/ml and are readily phagocytosed by macrophage cells in vitro as well as in vivo by alveolar macrophage. This study provides a framework for future design of inhalable β-glucan based drug carriers as a host-directed, targeted drug delivery system against Pulmonary TB.
Article
Rifabutin loaded Inhalable β-glucan micro particle based drug delivery system for pulmonary TB
https://doi.org/10.21203/rs.3.rs-4151640/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 16 Jul, 2024
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Anti- TB Drug Delivery
Alveolar Macrophages
Rifabutin
β-Glucan Microparticles
The objective of this study is to develop rifabutin-loaded inhalable microparticles derived from yeast for an efficient drug delivery system. The biophysical properties of particle including size, shape, surface morphology, cytotoxicity, and in-vivo/in-vitro phagocytic uptake, as well as aerodynamic characteristics following a spray drying process, were investigated. Comprehensive characterization demonstrates improved size, shape, and thermal stability of the particles. Moreover, the rifabutin-loaded microparticles exhibit enhanced Inhalability as compared to blank particles. In vitro and in vivo phagocytic uptake assessments reveal the particles' susceptibility to uptake by human macrophage cells and adequate biocompatibility. This research lays the foundation for potential applications in host-directed anti-TB therapy for managing pulmonary tuberculosis. Through the development of these innovative microparticles, a more effective and targeted treatment approach for tuberculosis.
Tuberculosis [TB] still persists as one of the most common infectious diseases in the world, causing 10 million deaths worldwide annually [1]. despite of the clinical advancement in medical sciences, TB exists as an alarming threat for mankind due to the emergence of multidrug resistance [MDR]/ extensive drug resistance [XDR] and total drug resistance [TDR], further increasing the global bio burden of this fatal disease and worsening the situation [2, 3, 4]. The current conventional oral therapy is inefficient as it often fails to completely cure patients, due to multiple reasons such as poor drug solubility, less bioavailability to the site of infection, drug degradation as well as toxicity and prolonged treatment duration, leading to non-compliance issues. In this context, recent advancements in the form of biocompatible and biodegradable nano- or micro-particle based targeted drug delivery systems forms a promising approach for the mitigation of these issues [5, 6]. β-glucans are one of the most abundant polysaccharides found in the cell walls of bacteria, fungi, cereals, etc., and consist of long glucose chains with β 1-3, 1-6 glycosidic linkages. These have been granted the Generally Regarded As Safe [GRAS] status by the USFDA and have also been recommended by European Food Safety Authority [EFSA] for boosting the immune function [7]. Recently, the yeast derived glucan particles have gained attention as biodegradable and biocompatible drug delivery systems that are porous in nature and thus allow encapsulation of various molecules including anti-TB drug within these particles [8, 9, 10]. These particles also serve as Pathogen Associated Molecular Patterns [PAMPs], and are recognized by the macrophage receptors including Dectin-1, CR-3 and FC-γ [11]. They are thereby efficiently taken up by macrophage and assist in the release of the encapsulated drug in targeted manner within the macrophages [12].
Pulmonary drug delivery in form of inhalable particles offers several advantages such as improved drug bioavailability, enhanced patient compatibility, reduced systemic side effects and prolonged drug action over conventional oral therapy [13, 14, 15]. The size of the particles plays an important role in deep lung delivery, as well as in their amenability to uptake by resident alveolar macrophages, leading to intracellular release of the encapsulated drug molecules [16, 17,]. Studies reveal that for efficient pulmonary drug delivery and deposition during inhalation, the mass median aerodynamic diameter [MMAD] of particles should be less than 5 µm, with Gravitational Standard deviation [GSD] values between 0.5 - 2.5 µm [18, 19,20].
Spray drying is generally used for the preparation of the inhalable microparticles; it consists of the three steps: atomization, drying, and collection [21, 22, 23]. L-leucine addition during particle preparation is reported to improve the dispersibility of particles during spray drying and the aerosolization and physical stability of microparticles, due to its enrichment on particle surfaces and intermolecular interactions with drugs [24, 25, 26]. The objective of this study was to develop β-glucan particles loaded with rifabutin and assess their inhalable characteristics using both in-vivo and in-vitro method for the betterment of pulmonary TB management. The development of this Rifabutin-loaded Inhalable β-glucan Micro Particle-based Drug Delivery System represents a significant advancement in TB therapy, offering a targeted and efficient approach to combatting this infectious disease. Through further research and clinical trials, this innovative system holds the potential to revolutionize the treatment of pulmonary TB, ultimately improving patient outcomes and reducing the global burden of this deadly disease [27, 28].
Reagents & solutions: For the extraction of β-glucan, baker’s yeast was purchased from a local shop in Lucknow. Anti TB drug, Rifabutin was procured from Lupin pharmaceuticals, and other chemicals such as l-leucine, acetone, isopropyl alcohol, HCl [analytical grade], DMSO were purchased from Merck. All cell culture reagents including FBS, RPMI-1640, antibiotic-antimycotic mixtures were purchased from Gibco. PMA was purchased from Thermo fisher, rhodamine and DAPI from Sigma. All aqueous solutions were prepared in triple distilled water [TDW].
Cell culture: THP-1 human macrophage cells was procured from the National Centre for Cell Science [NCCS], Pune and all working reagents were prepared in Milli-Q water and working stocks were stored as per manufacturer instructions at -20oC for further processing.
Preparation of L-leucine added, Rifabutin loaded β-glucan particles: Glucan particles were prepared by slight modification of the alkaline acidic extraction [AAE] method as described previously [Upadhyay et al 2017], such that l-leucine was also used in addition, as an excipient for particle preparation. Briefly, baker’s yeast was suspended and stirred in 1M NaOH, heated at 60°C, followed by centrifugation at 200×g. Then the sediment was treated with acidic water and subsequently washed with alcohol and acetone, followed by spray drying in presence of l-Leucine [Table 1], to obtain white colored β-glucan particles. Rifabutin loading and alginate sealing of the particles was performed as previously described. For some experiments the fluorescence tagging of these particles by Rhodamine-B was done as previously described [10]. The overall methodology of particle preparation has shown in [Fig: 1].
Biophysical characterization
Particle size: Particle size was determined by Laser scattering [Malvern Mastersizer] and SEM as previously described [Sharma et al., 2001, Upadhyay et al., 2017]. Briefly, blank and drug-loaded particles mixed with an equal amount of SDS and suspended in 1ml Milli-Q water, were subjected to laser obscuration at a factor of > 10% for size determination by Laser scattering. These particles were also visualized by Scanning Electron Microscope [FEI Quanta 250] at an accelerated voltage of 15 kV.
Fourier Transform Infra-red [FTIR] spectroscopy: Biochemical analysis of the prepared particles was carried out by FT-IR spectrometer [Perkin Elmer], at a scanning range from 400 cm-1 to 4000 cm-1 for both blank and drug loaded particles.
Differential Scanning Calorimetry [DSC]: The thermal stability of blank and drug loaded particles was determined by Differential Scanning Calorimetry. For this [5 mg sample] were placed in an aluminum pan [TA Instruments Discovery model DSC-25, New Castle, DE.USA], which was then sealed and allowed the determination of glass transition Tg, melting point Tm, and enthalpy change ΔHm. Empty pan were used as a reference in the furnace, heating range of sample pan was from 30°C - 200°C and flow rate was kept at 5°C min-1, when required, using an oxygen flow rate of 10 cm 1 min-1 .
Quantification of Drug: Quantification of incorporated drugs within particles was carried out by HPLC with slightmodification of the protocol as reported by Muttil et al. [2007]. Briefly, we prepared the mobile phase by mixing acetonitrile: methanol [45:55], which was filtered through a 0.22 µm filter and pump flow rate was kept at 1 ml/min. RB was seen to be eluted at 8.5 min and correspondingly, the standard curves were generated in the concentration range of 2-12 µg. Rifabutin was extracted from the drug-loaded-alginate sealed GP suspended in 0.01 N HCl, and then diluted in mobile phase after filtration through a 0.22µm filter. The drug loading efficiency [LE] for RB within the particles was calculated as follows:
Evaluation of Aerodynamic behavior of Particles: Aerodynamic behavior of the particles was carried out from the Inhalation Toxicology Unit, Institute of Industrial Toxicological Research [CSIR-IITR] Lucknow. Flow property of particles was evaluated through multi-orifice Andersen cascade impactor [MOUDI Cascade Impactor 110, MSP Corporation, Shoreview, MN, USA]. Ten mg of blank and drug loaded particles were placed in the pump. The particles deposited at each stage were collected and the Mass Median Aerodynamic Diameter [MMAD] and Geometric standard deviation [GSD] of the particles was calculated as per deposition profile at different stages of impactor. All the samples were analyzed in a triplicate manner [29].
In-vivo phagocytic uptake of the particles: To investigate if these particles are inhalable or not, we performed in vivo inhalation of these particles in 4 week-old, 4-6 g BALB/c mice using the inhalation apparatus as described previously [17, 30,31]. Briefly, 10 mg fluorescent tagged RB-GP particles were kept in inhalation apparatus for inhalation by mice for 10-30 min. The Bronchoalveolar lavage [BAL] fluid collected from lungs of these mice was centrifuged for 5 min at 1000 rpm to obtain cells that were dispensed in 6 well plates for visualization under fluorescence microscope.
In-vitro phagocytic uptake of the particles: In vitro phagocytic uptake of the particles was analyzed using human macrophage THP-1 cells. 1×104 cells were seeded in each 96 well plates and adhered using [50 ng] PMA and plates were incubated for 48 hours for proper adherence. After that PMA containing media was discarded and washed with incomplete RPMI-1640 media and incubated for 24 hours with complete media. Subsequently, the cells were incubated with Fluorescent tagged particles for 10-30 min for particle uptake. After that cell were washed thrice with 1X PBS and visualized under Fluorescence Microscope [EVOS FLoid Cell Imaging Station Model No: K1115-FLD2-084].
In-vitro cell viability assay: The viability of THP-1 cells post-exposure to the aforementioned formulation was evaluated by MTT assay. Briefly, 1×104 cells were seeded in each 96 well plate and adhered by 50 ng Phorbol Myristic Acid [PMA]. Cells were treated with different concentration of particles [10 - 80 μg/ml] and plates incubated overnight. Thereafter, 100 μl MTT dye was added for 3-4 h, after that 100 μl DMSO was added to dissolve the formazan and plates were read at 570 nm using ELISA Plate reader. Cell viability was calculated using the following formula:
Statistical analysis: Statistical data analysis was done by graphpad software by applying the One-way analysis of variance [ANOVA]. All data has been taken in a triplicate manner and expressed as mean ± standard deviation. The data was considered to be significant if p < 0.05.
Particle size distribution and morphology: The average particle size distribution for both the blank and drug loaded particles, was found to be in the range between 1 to 5 µm. The drug loaded particles were found to have unimodal particle distribution [poly dispersed], with a higher fraction [88%] of particles in the 1–5 µm range, and a very small fraction of the particles [5–10%] was found above the 5 µm [Fig: 2 A, B]. The size and surface morphology of these particles were also examined by SEM, which revealed that while the blank particles were collapsed and shriveled, with an uneven surface, the drug loaded particles were spherical in nature [Fig: 2 C, D, E, F]. The hollow cavity within blank particles and drug encapsulation within these particles was also visualized under a bright field microscope [Fig: 2 G, H].
FT-IR spectra of particles: The FT-IR spectra of these particles confirmed the presence of beta-glucan and Rifabutin specific functional groups within the prepared formulations Regarding the glucan specific functional groups, the absorbance peak at 928–1200 cm–1, due to C-C and C = O stretching vibrations confirmed the presence of pyranoids rings, indicating polysaccharide nature of the particles. The absorption peak at 3400.06 cm–1 represents the hydroxyl group of OH stretch, while the signals at 1215.72cm–1 indicate C-H stretch and CH2OH stretch. The absorption peak at 850.84 cm− 1 is characteristic of β-glycosidic bonds, i.e. [C1–H] deformation mode, and, thereby, indicates the presence of β-glucans. Absorption peak at 760.71 cm–1 shows presence of α-glycosidic bond. The peaks 1628 cm–1 and 1406 cm–1 indicate presence of a small fraction of mannosylated residual proteins [32, 33]. The FTIR spectra of Rifabutin-loaded glucan particles revealed Rifabutin specific functional groups with peaks at 1064.67 cm–1 indicating the presence of secondary alcohol and, peaks at 1253.91 cm–1 for the carbonyl stretch of the C = O groups, at 1718.88 cm–1, 1638.67 cm–1 vibration for the C = O stretching. Absorbance peaks at 3200.36 cm–1, 1605.24 cm–1 represent the N-H stretch and N-H deformation 2nd amine. Other peaks at 1418.84 cm–1, 1380.95 cm–1 indicate the C-H deformation of CH2 group of rifabutin and at 1600.24 cm–1 for vibration of C = C stretch [34]. The FTIR spectra of drug loaded alginate sealed particles exhibit peaks that overlap with the peaks of glucan and rifabutin specific groups, thereby confirming no change in the chemical structure of rifabutin, and indicating no loss in biophysical property of Rifabutin during its encapsulation within glucan particles [Fig: 3].
Differential Scanning Calorimetry [DSC] Analysis
Differential Scanning Calorimetry was used to analyze the purity and thermal stability analysis of the particles that showed broad endothermic peaks from 50°C to 128°C corresponding to the breaking of molecular hydrogen bond and evaporation of residual water molecules from β-glucans. Drug loaded and sealed particles were found to have higher glass transition temperature in comparison to the standard and extracted glucan [Fig: 4].
|
Blank |
Inlet Temp (°C) |
Aspirator |
Outlet Temp (°C) |
Pump Flow rate (ml/min) |
Particle yield in (gram) |
Polymer Drug ratio (gram) |
Leucine conc. in (mg) |
||||||
|
Blank GP-A |
145°C |
50% |
45°C |
5ml/min |
3.48gm |
1:1 |
100mg |
||||||
|
Blank GP-B |
150°C |
50% |
50°C |
5ml/min |
4.08gm |
1:1 |
200mg |
||||||
|
Blank GP-C |
155°C |
50% |
55°C |
5ml/min |
4.48gm |
1:1 |
300mg |
||||||
|
Characteristics of Rifabutin Drug loaded (GPRB) β-glucan particle. |
|||||||||||||
|
Drug |
d0.1 (µm) |
d0.5 (µm) |
d0.9 (µm) |
Drug Loading Efficiency (%) |
MMAD (µm) |
GSD (µm) |
|||||||
|
GPRB-A |
0.210µm |
5.800µm |
19.315µm |
67% |
1.58 |
1.43 |
|||||||
|
GPRB-B |
-- |
-- |
-- |
68% |
2.07 |
1.81 |
|||||||
|
GPRB-C |
0.908µm |
1.413µm |
3.496µm |
70% |
1.02 |
1.63 |
|||||||
Particle Drug content: Drug content estimation by HPLC [Fig: 5] revealed a loading efficiency of 68–70% within GPRB formulations [Table: 1]. the drug loading efficiency of the formulation was seen to be greatly improved by increasing the Leucine content and ratio of Drug: Polymer: Leucine.
Aerodynamic behavior of Glucan Particles: The Mass Median Aerodynamic Diameter [MMAD], and GSD value of the blank and drug loaded particles was found to be 1.01 to 1.62 µm and 1.02 to 2.07 µm respectively [Table: 1]. The mass median diameter [MMAD] and geometric standard deviation [GSD] of the particles were calculated by sigmoid curve fitting of the values of cumulative mass percent of particles undersize deposited on the stages against the log10 values of effective cut-off diameter of the stages showing in. [Fig: 6 [A], [B].
In-vivo and in-vitro phagocytic uptake: We found that these particles have significant aerodynamic diameter as well as Gravitational sedimentation diameter almost 1–8 µm of particles making them suitable for inhalation. They have greater propensity to reach the lungs and get phagocytosed by alveolar macrophage cells isolated from BAL fluid. [Fig: 7]. It's clearly seen in phagocytosed cells with emission of red fluorescence which is indicated by white arrow in confirming that the particles are readily phagocytosed by alveolar macrophages in a time dependent manner[Fig: 8]. The rhodamine-B labeled particles were seen to be readily phagocytosed in vitro by human macrophage within 10 min of exposure [Fig: 9].
In-vitro cytotoxicity: The blank as well as drug loaded GP did not show any cytotoxicity to THP-1 cells post 24 hours exposure at 10 µg/ml concentration and percentage of the cell viability was found to be almost 90% upto a concentration of 50 µg/ml [Fig: 10]. Further slight decline in cell viability was seen at higher concentrations that were insignificant upto 80 µg/ml, indicating that the particles are fairly biocompatible in nature and thus safe for exposure to cells and tissues.
Conventional TB therapy requires administration of high dose of anti-TB drugs for prolonged duration, leading to non-compliance issues and the emergence of the multi-drug resistance. A large number of recent studies have shown that pulmonary drug delivery of particles in form of dry powder inhalations [DPI], allows delivering drugs directly to lungs, and results in local drug concentration much higher than that achievable by oral administration. As opposed to building up high drug concentrations in blood by conventional oral treatment, this facilitates targeting of anti-TB drugs to alveolar macrophage harboring mycobacteria, builds up high intracellular drug concentrations and thereby enhances drug efficacy [10]. Thus, a great deal of research has attempted to generate aerosols for inhalation and deposition to lungs, or design rational “smart” particles based on the biological information on barriers [21, 22, 23]. Glucan particles offer dual advantage as biocompatible and biodegradable immunomodulatory particles that boost the immune function during various immune challenges and also allow encapsulation of nano-sized molecules including drugs, proteins, DNA and RNA. Additionally, the glucan nature of these particles permits their receptor mediated endocytosis by macrophage. Our previous studies have shown that RB loaded glucan particles confer enhanced protection against M.tb. as compared to that by equivalent amount of free drug [10, 35]. Considering the potential of inhalable particles to reach alveolar macrophage and deliver drugs, we attempted to develop an inhalable Rifabutin loaded, β-glucan particle based delivery system for Pulmonary TB, the most common form of TB. Spray drying technology has been extensively used to prepare inhalable, uniform structured particles in a narrow size distribution. The preparation of RB-loaded glucan particles by spray drying process generated by the cyclone, yielded uniform, hollow spherical particles that shriveled in shape after drying. The delivery of drug loaded particles to deep lungs mainly depends on the flow properties of the particles. Inter-particle agglomeration can hinder the alveolar penetration attributed to static charges, even though spray dried micronized particles have small aerodynamic diameters. The addition of amino acid leucine has been documented to enhance the flow property of particles by reducing inter-particle cohesion force and lowering the agglomeration issue of particles [18, 36, 37]. Additionally, conventional DPI additives such as leucine and lactose generally improve the properties of formulations such as uniformity and pulmonary penetration. The uniform, poly dispersed, and better yield of RB loaded Glucan particles is therefore attributed to the addition of leucine during the preparation process. The presence of active excipients like leucine and alginate layer on the drug loaded particles is expected to enhance their thermal behavior as compared to the extracted and standard glucan particles [38]. Drug loaded alginate sealed β-glucan particles exhibited significant higher thermal stability as compared to other formulations due to presence of the triple helical structure of glucan particles which is assumed to provide better thermal strength [38]. Particles subjected to temperature above Tm are known to undergo oxidation and thermal degradation. The DSC data also showed that melting temperature [Tm] of the standard and extracted glucan are almost the same. The DSC and FTIR data thereby confirmed that the extracted glucan particles do not have any impurity and biochemical discrepancy. The particles with smaller diameter size have a higher chance to reach deep in the lungs, extra fine particles are usually exhaled out during respiration. The optimum size range of the particles reported to readily reach the deep lung area lies in the range of 0.5 to 6.0µm diameter [20].
The formulations have been found to have sufficient aerodynamic property as showed in mass deposition. According particle deposition on each stage of the cascade impactor, deposition profile of particle and MMAD data has shown particles appear to be suitable for deep lung [38, 39]. The size and shape of particles indicate that these are amenable for receptor mediated uptake by macrophage. Our in vitro studies have shown these particles are fairly biocompatible in nature and are efficiently phagocytosed by macrophage. In the past few years many of the researchers has prepared the micro particle based drug delivery system for pulmonary drug with liposomal RFB formulation prepared with dipalmitoyl phosphatidylcholine:dipalmitoyl phosphatidylglycerol [DPPC:DPPG] the immunotherapeutic efficacy of these formulation were compared with mice treated with free RFB, mice treated with the DPPC: DPPG RFB formulation exhibited lower bacterial loads in the spleen [5.53 log[10] vs. 5.18 log[10]] and liver [5.79 log[10] vs. 5.41 log[10]] [39, 40, 41, 42, 43, 44]. In the lung, the level of pathology was lower in mice treated with encapsulated RFB. These results suggest that liposomal RFB is a promising approach for the treatment of extra-pulmonary TB in human immunodeficiency virus co-infected patients [45, 46, 47].
Since optimizing aerosol deposition is important but insufficient for pulmonary delivery, multifunctional “smart” particles are desirable to meet the higher delivery requirements. However, there is still a long way before these particles be available in market. Deeper understanding of the obstructive lung diseases is needed for particle development. Moreover, there are several issues needed to be clarified before the designed particles entering into the clinical use, such as safety of excipients, potential toxicity of nanoparticles on the immune system and differences of lung barriers between animals and human. Comprehensive studies are need in these fields in the future [48, 49, 50, 51]. Thus, this formulation holds a promising prospective for carrying large doses of drug, providing targeted delivery with the minimal side effects. Our further interest to evaluate the intra cellular immune response mediated by these particles within mycobacterium infected host macrophage cells.
Animal Ethical approval: Animal experimentation performs in this study as per study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Institutional Animal ethical committee approval obtains from Chhatrapati Shahu Ji Maharaj University Kanpur [IAEC/UIP/August 2023/025]. For experimentation we used healthy Balb/c mice and maintained in institutional animal house under 12hrs dark/light condition along with bottle of water and feeding pellet in animal cage with optimum parameter. For the isolation of Broncheo-alveolar lavage mice euthanized disposed as per biomedical waste management facility, Chhatrapati Shahu Ji Maharaj University, Kanpur.
Funding: The financial support received from Department of Science & Technology, Science & Engineering Research Board [DST-SERB] as grant number [DST-SERB/CRG/2019/007035/BHS] is gratefully acknowledged. The DST-FIST [SR/FST/LS-1/2017/13/[C]] sponsored Department of Biosciences and also acknowledged for providing the manuscript no. [IU/R&D/2023-MCN0001913].
Acknowledgement: The authors are thankful to the Deanship of Scientific Research, King Khalid University, Abha, Saudi Arabia, for financially supporting this work through the small Research Group Project under Grant no. [R.G.P.1/226/44]. I would like thanks to Dr. Amit Misra (senior scientist) Pharmaceutics & Pharmacokinetics division, (CSIR-CDRI) Lucknow, for their kind help and support.
Conflict of interest: The authors have declared not ant type of conflict of interest within manuscript.
Credit of Authorship: Conceptualization of Rough draft, Data collection, editing, artwork done by FA, and examination RS and MK, TKU, MS and IA, Aerodynamic experiments study helped by JS, SEM HPLC, DSC, helped by DS,SS. And final editing suggestions, revisions done by RS and MK.
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No competing interests reported.