Functionalised nanoparticles for antimicrobial therapy

Abstract

A polymeric macrophage-targeting nanoparticle delivery system comprising: an outer chitosan shell functionalized with 1,3-β-glucan and optionally at least one active compound; a hydrophobic polymer core comprising an immunomodulating agent and optionally at least one active compound; wherein where there is no active compound provided in the outer chitosan shell, there is an active compound provided in the hydrophobic polymer core and wherein where there is no active compound provided in the hydrophobic polymer core there is an active compound provided in the outer chitosan shell.

Description

[0001] FUNCTIONALISED NANOPARTICLES FOR ANTIMICROBIAL THERAPY

[0002] FIELD OF THE INVENTION

[0003] This invention relates to polymeric macrophage-targeting nanoparticles for use in antimicrobial therapy, a method of preparation of the polymeric macrophage-targeting nanoparticles, and applications thereof.

[0004] BACKGROUND TO THE INVENTION

[0005] Macrophages are known to combat bacteria, viruses and other microorganisms through various phagocytosis-related antimicrobial responses. Recently, much effort has been expended to create novel / improved therapeutics by regulating host-directed therapies (HDTs) to microorganisms, particularly antibiotic-resistant bacterial infections, through manipulation of the macrophage antimicrobial response route (Paik et al., 2019, Khoza et al., 2022, Kolloli and Subbian, 2017).

[0006] In contrast to traditional therapy, HDTs target pathways disturbed by the pathogen without interfering with the pathogen to boost host defence systems. Autophagy is an intracellular catabolic process that aids in maintaining homeostasis or removing invading pathogens through a lysosomal degradation process. The activation of autophagy by various drugs or agents may represent a promising antimicrobial treatment strategy, including for viruses and even for drug-resistant bacteria such as drug resistant Mtb (Paik et al., 2019).

[0007] There is a great variety of nanoparticles which have been widely utilised to target many different drug molecules or antimicrobial agents to cells, including macrophages. Depending on the drug molecule or antimicrobial agent and the pathogen being targeted, different nanoparticles have been considered and many different modifications of various nanoparticles have been investigated. It would be an advantage to develop a functionalised nanoparticle that could be targeted against multiple potential pathogens, including viruses and bacteria, particularly drug resistant strains, wherein the functionalised nanoparticle enhances the effect of encapsulated drug molecules and / or antimicrobial agents. It would be useful if the functionalised nanoparticle could additionally contribute to the antimicrobial response through macrophage-targeting to initiate a host-directed response.

[0008] SUMMARY OF THE INVENTION

[0009] According to a first aspect of the invention, there is provided a polymeric macrophage- targeting nanoparticle delivery system comprising: an outer chitosan shell functionalized with 1 ,3-β-glucan and optionally at least one active compound; a hydrophobic polymer core comprising an immunomodulating agent and optionally at least one active compound; wherein where there is no active compound provided in the outer chitosan shell, there is an active compound provided in the hydrophobic polymer core and wherein where there is no active compound provided in the hydrophobic polymer core there is an active compound provided in the outer chitosan shell.

[0010] The hydrophobic polymer core may be comprised or consist of a polymer selected from polylactide or polylactic acid (PLA), polyglycolide or polyglycolic acid (PGA), polycaprolactone, polydioxanone and a range of their copolymers. In particular, the hydrophobic polymer core is a polycaprolactone core.

[0011] It is known that polycaprolactone is an aliphatic polyester, a class of synthetic, biodegradable, and biocompatible polymers known for their slow degradation rate and good mechanical properties. Aliphatic polyesters contain ester functional groups in their backbone. The "aliphatic" designation indicates that these functional groups are part of an open-chain hydrocarbon. Polycaprolactone is classified with other biodegradable polyesters, such as polylactic acid (PLA), polyglycolic acid (PGA), polydioxanone or their co-polymers which would be equally expected by one skilled in the art to be possible alternatives to polycaprolactone in the hydrophobic polymer core.

[0012] An immunomodulating agent in the context of this application means to provide modulation (regulatory adjustment) of the immune system from natural and human- induced forms and refers to inducing, amplifying, attenuating, or preventing a full immune response (or intermediatory response) according to the therapeutic goals in the context of host-directed (or augmented) therapy.

[0013] The immunomodulating agent may be selected from cholecalciferol (VITD3), vitamin A, vitamin C, vitamin E, and β-carotene, dehydroascorbic acid, metformin, as well as microelements such as zinc, selenium, iron, omega-3 fatty acids, and live active probiotic bacteria. In particular, the immunomodulating agent is VITD3.

[0014] It is to be appreciated that the polymeric macrophage-targeting nanoparticle delivery system of the invention works on a synergistic approach. VitD3 is one possible immunomodulator shown to exponentially enhance or act in conjunction with β-glucan targeting, stimulating the immune system to target and fight infectious diseases by binding to cell-surface receptors on immune cells like macrophages and neutrophils. However, the polymeric macrophage-targeting nanoparticle delivery system of the invention carrying any immunomodulator capable of providing host-directed therapy would likewise be expected to work in tandem with fJ-glucan targeting.

[0015] The polymeric macrophage-targeting nanoparticle may have a size of between about 100 nm to about 200 nm, or from about 120 nm to about 190 nm, or any size range therebetween.

[0016] The polymeric macrophage-targeting nanoparticle may comprise more than one active compound. The at least one active compound may be hydrophobic or hydrophilic. One active compound may be hydrophobic and the other hydrophilic. In particular, the at least one additional active compound may be hydrophobic.

[0017] The at least one active compound may be a compound active against a macrophage- targeting pathogen including a bacterium, a virus, a fungal pathogen and the like. The at least one active compound may be an antibiotic, antifungal, antiviral, antimalarial, anti-TB drug, antiretroviral, or the like. In particular, the active compound is Rifampicin (RIF).

[0018] The polymeric macrophage-targeting nanoparticle may be formulated for delivery, including by oral, transdermal, buccal, pulmonary, nose-to-brain, or intravenous drug delivery. For example, the polymeric macrophage-targeting nanoparticle may be comprised in a hydrogel or thermogel delivery system. More particularly, the hydrogel or thermogel delivery system comprising the polymeric macrophage-targeting nanoparticle of the invention may be adapted to provide prolonged and controlled release properties for the compounds to be delivered.

[0019] According to a second embodiment of the invention there is provided a pharmaceutical composition comprising a polymeric macrophage-targeting nanoparticle of the invention for macrophage-targeted delivery of at one active compound.

[0020] The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient.

[0021] According to a third embodiment of the invention there is provided a method for preparing the polymeric macrophage-targeting nanoparticle of the invention comprising the following steps: a) providing a hydrophobic polymer solution in a polar, aprotic solvent and further comprising an immunomodulatory agent and optionally an active compound as the organic phase; b) providing a solution comprising polyvinyl alcohol (PVA), chitosan oligosaccharide lactate, and optionally an active compound; c) adding, dropwise, the organic phase of a) to the solution of b) under stirring to yield a solution; d) sonicating the solution of c), followed by stirring to evapourate excess polar, aprotic solvent to form a solution of nanoparticles comprising an outer chitosan shell and optionally at least one active compound, and a hydrophobic core comprising the immunomodulatory agent and optionally at least one active compound, wherein where there is no active compound provided in the outer chitosan shell, there is an active compound provided in the hydrophobic polymer core and wherein where there is no active compound provided in the hydrophobic polymer core there is an active compound provided in the outer chitosan shell; e) collecting the solution of nanoparticles of d) for 1 ,3-β-glucan functionalisation; f) incubating the solution of nanoparticles with a 1 ,3-β-glucan solution thereby to attach the 1 ,3-β-glucan to the outer chitosan shell thereby to generate the polymeric macrophage-targeting nanoparticles; g) purifying the polymeric macrophage-targeting nanoparticles by centrifugation and washing; and i) optionally lyophilising the purified polymeric macrophage-targeting nanoparticles.

[0022] The hydrophobic polymer may be comprised or consist of a polymer selected from polylactide, polyglycolide, polycaprolactone, polydioxanone and a range of their copolymers. In particular, the hydrophobic polymer is polycaprolactone.

[0023] The polar, aprotic solvent may be selected from the group comprising dicholoromethane (DCM), acetone, ethanol, or a mixture thereof. In particular, the polar, aprotic solvent may be DCM.

[0024] The immunomodulating agent may be selected from cholecalciferol (VITD3), vitamin A, vitamin C, vitamin E, and β-carotene, dehydroascorbic acid, metformin, as well as microelements such as zinc, selenium, iron, omega-3 fatty acids, and live active probiotic bacteria. In particular, the immunomodulating agent is VITD3.

[0025] The at least one active compound may be a compound active against a macrophage- targeting pathogen including a bacterium, a virus, a fungal pathogen and the like. The at least one active compound may be an antibiotic, antifungal, antiviral, antimalarial, anti-TB drug, antiretroviral, or the like. In particular, the active compound is Rifampicin (RIF).

[0026] According to a fourth embodiment of the invention, there is provided a method of administering a pharmaceutically active compound to a subject in need thereof, the method comprising a step of administering polymeric macrophage-targeting nanoparticles or a pharmaceutical composition of the invention to the subject.

[0027] According to a fifth embodiment of the invention, there is provided polymeric macrophage-targeting nanoparticles or a pharmaceutical composition of the invention for use in a method of administering a pharmaceutically active compound to subject in need thereof.

[0028] The administering may comprise oral, transdermal, buccal, pulmonary, nose-to-brain, or intravenous drug delivery of the polymeric macrophage-targeting nanoparticles of the invention. For example, the administering may comprise delivery of a hydrogel or thermogel delivery system comprising the polymeric macrophage-targeting nanoparticle. More particularly, the hydrogel or thermogel delivery system comprising the polymeric macrophage-targeting nanoparticle of the invention may be adapted to provide prolonged and controlled release delivery of the pharmaceutically active compound.

[0029] DESCRIPTION OF FIGURES

[0030] The invention will now be described in more detail with reference to the following non- limiting example hereunder, and the accompanying drawing.

[0031] In the drawing:

[0032] Figure 1 shows a graphic illustration of generated particle size intensity profile and apparent particle zeta potential of (a) CS-PCL-VITD3 and (b) GLU-CS-PCL-VITD3 nanoparticle

[0033] Figure 2 shows a graphic illustration of generated particle size intensity profile and apparent particle zeta potential of (a) CS-PCL-RIF and (b) GLU-CS-PCL-RIF nanoparticles;

[0034] Figure 3 shows SEM images of (a) unloaded CS-PCL, (b) drug-loaded CS-PCL and (c) β-1 ,3-Glucan surface functionalised CS-PCL nanoparticles and their TEM images, (d), (e) and (f) respectively at different scales; Figure 4 shows FTIR spectra of a) excipient pristine polymers and formulated nanoparticle (CS-PCL) and b) and (b) formulated nanoparticle, excipient drugs / bioactive and drug-loaded nanoparticles (CS-PCL-VITD3 / RIF) showing various molecular composition and transition upon polymer hybridisation respectively;

[0035] Figure 5 shows FTIR spectra of pristine 1 ,3-beta-glucan (Glu), functionalised (Glu-CS-PCL) and non-functionalised (CS-PCL) nanoparticles showing various molecular composition and transition upon surface functionalisation of CS-PCL NPs;

[0036] Figure 6 shows XRD diffractograms of (a) all excipient materials, (b) formulated NPs with and without drug encapsulation and (c) pristine 1 ,3-beta-glucan, functionalised and non-functionalised nanoparticles;

[0037] Figure 7 shows DSC thermograms of (a) Formulated nanoparticles (CS-PCL) and all raw excipient polymers, (b) Formulated nanoparticles and excipient drug / bioactives, and (c) 1 ,3-beta-glucan and Glucan functionalised CS-PCL NPs;

[0038] Figure 8 shows In vitro VITD3 release profile from a) CT-PCL-VITD3 and b) GLU- CS-PCL-VITD3 nanoparticles at pH 4.8, 6.8 and 7.4, at 37°C for 48 hours (n = 3, mean ± SD);

[0039] Figure 9 shows In vitro RIF release profile from a) CT-PCL-RIF and b) GLU-CS- PCL-RIF nanoparticles at pH 4.8, 6.8 and 7.4, at 37°C for 48-hours (n = 3, mean ± SD);

[0040] Figure 10 shows MTT assay measurement of the viability of RAW 264.7 murine macrophage cell after 48-hour treatment with free RIF, VITD3, CS-PCL and VITD3 / RIF CS-PCL nanoparticles at different concentrations (n = 3, mean ± SD); and

[0041] Figure 11 shows the relative Fluorescence Units (RFU) of Reactive Oxygen Species (ROS) generation in RAW 264.7 macrophage cells after 24-hour treatment with (a) 36 μg / mL of RIF pristine drug solution and 145 μg / mL of different RIF nanoparticle solutions, (b) 36 μg / mL of VITD3 pristine drug solution and 145 μg / mL of different VITD3 nanoparticle solutions. The concentration of TNF-α in RAW 264.7 macrophage cells following 24-hour treatment with (c) 145 μg / mL and 36 μg / mL of RIF nanoparticles and RIF pristine drug solution and (d) 145 μg / mL and 36 μg / mL of VITD3 nanoparticles and VITD3 pristine drug solution. Data is presented as mean ± SD (n = 3). Statistical significance between groups is indicated as follows: n.s. (not significant, p > 0.05), *** (very significant, p < 0.001) and ** (highly significant, p < 0.01);

[0042] Figure 12 shows the intracellular survival of M. tuberculosis H37Rv in macrophages. Activated THP1 cells were infected with H37Rv bacterial cells and treated with seven different formulations. Rifampicin was included as the control. Survival was monitored by enumerating colony-forming units (CFU / ml) after 5 days of incubation. Data is presented as mean ± SD (n = 3). Statistical significance compared to the control is indicated as ** (Highly significant, p < 0.05), * (significant, p > 0.05; and

[0043] Figure 13 shows MTT assay measurement of RAW 264.7 murine macrophage cell viability after 48-hour treatment with (a) pristine β-1 ,3-glucan and β-1 ,3-glucan functionalised nanoparticles at different concentrations (n = 3, mean ± SD).

[0044] DETAILED DESCRIPTION

[0045] This invention relates to β-glucan-functionalised polymeric-chitosan nanoparticles for use in antimicrobial therapy, a method of preparation of the β-glucan-functionalised polymeric-chitosan nanoparticles, and applications thereof.

[0046] The following description of the invention is provided as an enabling teaching of the invention, is illustrative of the principles of the invention and is not intended to limit the scope of the invention. It will be understood that changes can be made to the embodiment / s depicted and described, while still attaining beneficial results of the present invention. Furthermore, it will be understood that some benefits of the present invention can be attained by selecting some of the features of the present invention without utilising other features. Accordingly, those skilled in the art will recognise that modifications and adaptations to the present invention are possible and can even be desirable in certain circumstances, and are a part of the present invention.

[0047] EXAMPLE The Applicant has developed a 1 ,3-β-glucan-functionalised polymeric-chitosan nanoparticle comprising VITD3 capable of synergistically enhancing the efficacy of additional drug molecules or antimicrobial agents encapsulated therein together with the additional ability to induce a host-directed response through macrophage- targeting.

[0048] In this exemplary example, a synergistic enhancement of the 1 ,3-β-glucan- functionalised polymeric-chitosan nanoparticle was demonstrated by the Applicant through its application in the treatment of TB together with encapsulated Rifampicin. These nanoparticles were evaluated for their cytotoxicity against RAW macrophage cell lines. Furthermore, these nanoparticles' minimum inhibitory concentrations (MIC) were determined against both Mycobacterium tuberculosis (mTB) susceptible strain H37Rv and a resistant strain MDR11 .

[0049] 1. Background

[0050] With an estimated 10.4 million new cases and an average annual mortality rate of 1.7 million, TB remains a significant global health concern, especially in developing countries. Before the SARS-CoV-2 pandemic, it was the leading cause of mortality from a single infectious agent, with nearly one-third of the world's population infected with latent Mycobacterium tuberculosis (LTBI) (Khoza etal., 2022, Kolloli and Subbian, 2017, Mphaphuli et al., 2023). HIV coinfection, the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains, and the coexistence of other chronic conditions like diabetes all contribute to an acceleration of the morbidity and mortality rate caused by TB (Kolloli and Subbian, 2017).

[0051] After inhalation, Mtb enters the alveolar space, where it is bathed in alveolar lining fluid (ALF) and directly touches the soluble lung mucosa components before interacting with the cellular compartment, mainly alveolar macrophages (AM) and other immune cells (Allue-Guardia et al., 2021). The current TB treatment regimen consists of a six- month regimen that includes a two-month intensive phase in which patients receive four first-line antibiotics [isoniazid (INH), ethambutol hydrochloride (EMB), and pyrazinamide (PZA)], followed by a four-month continuous phase in which patients receive rifampicin (RIF) and INH (Maphasa et al., 2020, Allue-Guardia et al., 2021). Despite all the current chemotherapeutics, the global tuberculosis epidemic has worsened due to HIV co-infection, the absence of a viable vaccination, the length of treatment, drug toxicity, and the rise of multidrug-resistant (MDR) bacteria. In addition, the expensive, time-consuming anti-TB medication for cases requiring multiple drugs has adverse side effects that make it difficult for patients to adhere to their treatment regimen. There is therefore an urgent need for innovative new therapies, particularly against drug-resistant TB.

[0052] Vitamin D3 is one of the most researched autophagy mediators / activators against TB due to the link between vitamin D3 insufficiency, TB susceptibility, and its ability to promote autophagy and the synthesis of antimicrobial peptides (Lee and Bhakta, 2021).

[0053] Chitosan and it’s derivatives are natural antimicrobial polymers that are known to kill microbes including bacteria and viruses either directly or through the neutralisation of negative charges on the microbial surface. β-Glucan particles (GP) are polymeric carbohydrates derived from yeast that have been demonstrated to be useful for macrophage-targeted drug delivery and promoting immunomodulation. The functionalization of nanoparticles with 1 ,3-β-glucan notably increases their recognition and macrophage uptake through Dectin-1 receptors, promoting enhanced phagocytosis and targeted drug delivery (Dube et al., 2014).

[0054] 2. Materials and Methods

[0055] 2.1 . Materials

[0056] Polycaprolactone (Mn80,000), chitosan oligosaccharide lactate (CS, Mn5,000 Da), Cholecalciferol (Vitamin D3, MW; 384.64), Rifampicin (RIF, MW;822.94), β-1 ,3-Glucan from Euglena gracilis, poly (vinyl alcohol, 87-90% hydrolysed, MW; 30 000-70 000), and phosphate-buffered saline (PBS) were all purchased from Sigma-Aldrich (St. Louise, MO, USA). Dichloromethane (DCM) and methanol were procured from Merck (Pty Ltd., South Africa). RAW 264.7 cell line was procured from Separation Scientific Pty Ltd (Johannesburg, SA). All Elisa kits were purchased from ThermoFisher Scientific (LTC Tech South Africa Pty Ltd, Roosevelt Park) and Merck (Pty Ltd., South Africa). All chemicals were of analytical reagent grade and used without prior modification. Double deionised water (DDW) was used for all the preparations.

[0057] 2.2. of functionalised and non-functionalized

[0058] Cholecalciferol (VITD3) and RIF-loaded nanoparticles were prepared following a modified single emulsion method (TripathL,, et... aL, 2010) . Briefly, 20 mg / ml of polycaprolactone (PCL, Mn 80,000 Da) solution in DCM containing 2.5 mg / ml of either VITD3 or RIF was added dropwise to a 0.1 % w / v polyvinyl alcohol (Mn 31 ,000 Da) solution in double-distilled water (DDW) containing 2 mg / ml of chitosan oligosaccharide lactate (CS, Mn 5, 000 Da) under stirring at 1100 rpm at room temperature. The resulting solution was then probe sonicated for 20 min (in a pulsed manner, 40% intensity) over an ice bath. The solution was left stirring (1100 rpm) overnight at room temperature to evaporate excessive DCM. Nanoparticles were then collected through centrifugation at 12000 rpm for 20 min, followed by washing with DDW and lyophilised for 20 hours at temperature between -30 °C and 25 °C under vacuum collector pressure of 1.030 mBar. For functionalisation, a modified method by Dube et al. 2014 was followed (Dube et.al..2014). Briefly, 40 mL of nanoparticle solution was incubated with 250 μg / mL of 1 ,3-β-glucan solution in DDW for 24 hours to make a final concentration of 25 μg / mL of 1 ,3-β-glucan. Unattached 1 ,3-β-glucan was removed by centrifugation (5000 rpm for 5 minutes), followed by washing with DDW and lyophilisation.

[0059] 2.3. of size distribution zeta and index

[0060] The average hydrodynamic particle size, zeta potential, and polydispersity index (PDI) of encapsulated, unencapsulated, functionalised, and non-functionalized nanoparticles were measured using Malvern ZetaSizer Nano ZS (Malvern Instruments, Worcestershire, UK) at 25 °C. Briefly, each sample was concentrated (1 :9) with DDW, and 1 mL of each piece was placed in a disposable cuvette and measured. All measurements were done in triplicates.

[0061] 2.4. Evaluation and degree of B-1 ,3-qlucan functionalisation The entrapment efficiency of VITD3 and RIF encapsulated within non-functionalised (CS-PCL-VITD3 / RIF) and functionalised (Glu-CS-PCL-VITD3 / RIF) nanoparticles were determined by allowing each drug-loaded nanoparticle formulation to dissolve completely. Briefly, lyophilised nanoparticle formulation of known mass was dissolved in adequate PBS at pH 4.6 and left in an orbital shaker (25 rpm) incubator at 37 °C for 5 days to allow for complete dissolution. The resulting suspension was centrifuged at 5000 rpm for 5 min. The concentration of VITD3 and RIF in the clear supernatant was determined using an Implen Nanophotometer (IMPLEN Nanophotometer™, Implen GmbH, Munchen Germany) set at Amax = 265 nm and 335 nm, respectively, following generation of calibration curves for each drug (R2> 0.99). The percentage value of drug entrapment efficacy (%EE) and loading capacity (%LC) were determined using equations (1) and (2). The measurements were carried out in triplicates and presented as mean ± STD.

[0062] Aqrepresents the amount of drug measured by the Implen Nanophotometer (IMPLEN Nanophotometer™, Implen GmbH, Munchen Germany), and Tq represents the amount of the drug employed in the formulation.

[0063] Where Aqis the amount of drug measured by the Implen Nanophotometer, and W represents the weight of the nanoparticle formulation.

[0064] The density of β-1 ,3-Glucan on Glu-CS-PCL nanoparticles was evaluated using the modified phenol-sulfuric method (Granum and Myklestad, 2002, Nielsen. 2010, Taylor, 1995). Briefly, 100 μL of Glu-CS-PCL nanoparticles (5mg / mL) solution were dispensed into a 96-well plate. Concentrated sulfuric acid (150 μL) was ejected quickly onto the dispensed nanoparticles solution. The plate was shaken gently on a shaker to allow for homogeneous mixing of the solution before adding 5% (w / v) phenol solution (50 μL). The reaction was allowed to proceed for 1 hour on a shaker, permitting adequate reduction of β-1 ,3-Glucan. Non-functionalized CS-PCL nanoparticles were used as a control to account for polymer interference with the assay. The absorbance of standards and unknown samples were measured at a wavelength of 490 nm using a microplate reader (Perkin Elmer VICTOR X 3 2030 MultiLabel Plate Reader). The surface coverage of β-1 ,3-Glucan molecules on the functionalised nanoparticles was computed relative to the standard curve.

[0065] 2.5.

[0066] The morphological surface of the nanoparticles was determined by employing Scanning electron Microscopy (SEM) analysis (SIGMA VP, Zeiss Electron Microscopy, Carl Zeiss Microscopy Ltd; Cambridge, UK) and Transmission Electron Microscopy High-Resolution Transmission Electron Microscopy (HRTEM) version TECNAIF3OST-TEM. Dispersions of the nanoparticles were concentrated to approximately 1 :9 with DDW and sonicated for 30 seconds in a water bath. A drop of nanoparticle suspension for each sample was placed on a metallic sample stub and left overnight to dry before sputter-coating (x2) with gold-palladium (AuPd). Each sample was observed under different magnifications at an accelerated voltage of 20 kV for SEM. Similarly, TEM was employed in ascertaining the morphology of nanoparticles. A drop of the concentrated sample was mounted on the carbon-coated copper grid and air-dried under the fume hood before visualising it under the electron microscope.

[0067] 2.6. Evaluation of Fourier transform infrared

[0068] The FTIR spectra of different powdered samples were analysed for specific interactions between pristine polymers, drug / bioactive, and other fabricated nanoparticles using a 2000 ATR-FTIR (PerkinElmer 100, Llantrisant, Wales, UK) spectrophotometer fitted with a single-reflection diamond MIRTGS detector with the wavelength ranging between 4000-650 cm-1and a 4 cm-1resolution.

[0069] 2.7. X-i diffraction

[0070] The degree of crystallinity of the starting materials for all the fabricated nanoparticles, samples were evaluated by benchtop XRPD (Rigaku Mini Flex 600, Tokyo, Japan) diffractometer fortified with CuKa radiation at 15 mA and 40 kV. The 20 scan range was selected between 10-90 degrees at a scan rate of 10 degrees / minute. These diffraction analyses allowed for observing the degree of crystallinity of the fabricated nanoparticles. 2.8. Evaluation of the thermal of the

[0071] The fabricated nanoparticle and pristine polymers' thermal stability and decomposition temperature were evaluated using a Thermogravimetric analyser (TGA) (PerkinElmer, TGA 4000, Llantrisant, Wales, UK). Samples of > 10 mg were placed in the TGA furnace, and the measurements were carried out under a nitrogen atmosphere with a heating rate of 10 °C / min from 30 °C to 900 °C. The changes in sample weight were recorded and evaluated as a function of temperature. The thermophysical properties of the same samples were determined using a differential scanning calorimeter (DSC) (Mettler Toledo, DSC, STARe System, Swchwerzenback, ZH, Switzerland). The samples with a mass range between 3-10 mg were sealed in aluminium crucibles and heated over 20 to 400 °C at a heating rate of 10 °C / min under continuous nitrogen purging. DSC curves were plotted as heat flow against temperature.

[0072] 2.9. In vitro release of VITD3 and RIF in formulated

[0073] The drug release profile of VITD3 / RIF in the nanoparticles was evaluated using a dialysis membrane approach at three different pH values (4.6, 6.8, and 7.4). Briefly, for the nanoparticle release profile, the lyophilised nanoparticle sample was weighted and dissolved in an appropriate volume of PBS at different pHs, as mentioned above. The dissolved samples were then loaded into a dialysis tubing membrane (SnakeSkin™, 3500 MWCO) and submerged into the relevant drug release media buffer of the corresponding pH and incubated (37 °C) in an orbital shaker (25 rmp) (YIHDER LM-530, YIHDER Co., Ltd., Taipei, Taiwan) until completion of release study. The dialysis membrane was used to facilitate only free VITD3 / RIF diffusion into the release medium for assay. Sampling (2 mL) was undertaken at different time intervals (t = 1 , 2, 4, 8, 12, 24, and 48 h). An equivalent amount of pre-warmed fresh media was replaced into the sample release media to sustain sink conditions. Nanophotometer was employed to quantify VITD3 and RIF at 265 and 335 nm, respectively, with each experiment, carried out in triplicate.

[0074] 2.10. In vitro of formulated

[0075] The effects of the formulated nanoparticle and pristine drug / bioactive on RAW 264.7 macrophage cell line metabolic activity and cytotoxicity were carried out using an MTT cell proliferation Kit I (Roche, Basel, Switzerland). Briefly, DMEM supplemented with 1 % penicillin-streptomycin and 10% fetal bovine serum was used to culture the cells at 37°C and 5% CO2(in a humidified incubator). After every 48-72 hours, the spent growth medium was replenished with fresh growth medium until the cells reached confluence. The cells at a density of 1x105per well were seeded in 96-well plates in triplicates. After 24 h, the adhered cells were treated with nanoparticle solution, pristine drug, and nanocomposite hydrogel solution (62.5-500 μg / mL) at differential concentration ranges. Subsequently, the cells were incubated for 48 hours in a humidified incubator at 37°C and 5% CO2. Following the incubation period, each well plate was then treated with 10 μL; 5 mg / mL of MTT solution and further incubated for 4 hours before being treated with 100 μL of solubilising agent (acid-isopropanol; 0.04 N HCI in isopropanol) to dissolve formed formazan crystals and subjected to further incubation overnight at same conditions as mentioned above. The absorbance of cell activity was then measured with a multimodal microplate reader (Victor X3, PerkinElmer, Waltham, MA, USA) at 570 nm with a reference wavelength of 620 nm. A parallel assay experiment comprised positive (cells treated with culture medium only), negative (cells treated with 4% v / v DMSO) control, and blank wells only containing solubilising agent and growth medium. The absorbance measurements were computed (mean ±SD) and used to calculate % cell viability using Eq. (3):

[0076] Atest is test absorbance, Ablankis blank, and Acontrolis a control absorbance in nm.

[0077] 2.11. Quantification of ROS, Pro-inflammatory, and Anti-inflammatory Cytokines in

[0078] RAW 264.7 Cells

[0079] ROS and cytokine levels in RAW 264.7 cells were quantified using kits per the manufacturers' protocols. ROS was measured with the Fluorometric Intracellular ROS Assay Kit (Sigma-Aldrich). TNF-α and IFN-γ were assessed using high-sensitivity ELISA kits (Thermo Fisher Scientific), while IL-4 and IL-10 were quantified with Instant ELISA Kits (Thermo Fisher Scientific). Data was normalized to untreated controls.

[0080] 2.12. Evaluation of Formulated Aqainst M. tuberculosis usinq the Broth

[0081] Microdilution Method All methods related to mycobacterial experimentation were conducted in accordance with the relevant regulations related to handling human specimens and guidelines for the growth of M. tuberculosis and handling of human specimens. All procedures were conducted in a Biosafety Level III laboratory, registered with the South African Department of Agriculture Forestry and Fisheries (registration number: 39.2 / NHLS- 20 / 010) and approved by the Institutional Biosafety Committee of the University of the Witwatersrand (approval number: 20200502Lab).

[0082] The anti mycobacterial activity of the seven nanoparticles formulations together with VITD3 and RIF against a drug susceptible Mtb strain (H37Rv) and a drug-resistant strain (MDR11 ) was investigated using the broth microdilution minimum inhibitory concentration method as previously described by Kana et al., with modifications. To each well of a sterile round-bottom 96-well plate, except in the first row, 100 μL of Middlebrook 7H9 media (Difco) containing 10% Middlebrook oleic acid-albumin- dextrose-catalase (OADC), 0.2 % glycerol and 0.5 % Tween 80 was added. The formulated nanoparticles and VITD3 were prepared in double distilled water to 1 mg / mL stock concentration. Stocks were diluted to a final concentration of 100 μg / ml in 7H9 media and 100 μL of each was added in duplicate to wells in the first row of the plate. Using a multichannel pipette, 50 μl of mixture was removed from the first row and added to the second row containing 50 μl of media, mixed several times before removing 50 μl and adding it to the next row. The dilution series was continued to the end of the plate and 50 μl was discarded from the last row. The positive drug controls, rifampicin, isoniazid and streptomycin, were prepared to final concentrations to provide a range from 3,2 μg / mL to 0.002 μg / mL, 6.4 μg / mL to 0.004 μg / mL, and 100 μg / mL to 0.04 μg / mL, respectively during the 2-fold serial dilution across the 96 well plate. An equivalent amount of DMSO was used to dissolve the nanoparticle formulations, and the anti-tuberculosis drugs was included together with wells containing cells only (uninfected) as controls. Mtb strains were grown in Middlebrook 7H9 broth at 37 °C for 3-4 days to an OD600nm = 0.5 - 0.8. The bacterial cultures were diluted 1 :500 in 7H9 medium, and 50 μl was added to each well. The plates were incubated for 14 days in a CO2 incubator at 37°C and scored visually using an inverted mirror for growth as pellets at the bottom of the well at 7 and 14 days. The last row, where no growth was observed, represented the MIC for the nanoparticle formulation. Wells in which growth could not be visually confirmed, 10 μl of Alamar Blue (Invitrogen) was added on day 14 and the plates incubated overnight before visually scoring for change in colour from blue (no growth) to pink (viable growth) using an inverted mirror.

[0083] 2.13. Intracellular killing of Susceptible Mtb, H37Rv in TH P-1 -activated Macrophages

[0084] THP-1 monocytes (Merck) were grown and maintained in RPMI-1640 media (Gibco) supplemented with 10% FBS, Sigma at 37°C, and 5% CO2for 7-10 days, after which viability was assessed by trypan blue exclusion staining. 1 ml of cells with a density of 2 x 105cells / ml were seeded in each well of a 48-well plate. THP-1 monocytes were differentiated into adherent, activated macrophages with 100 nM phorbol myristate acetate (PMA) (Sigma), 150 U of recombinant human gamma interferon (IFN-y) for 3 days at 37°C, 5% CO2. A culture of Mtb, H37Rv was grown in 7H9 media supplemented with 0.2% glycerol, 10 % OADC, and 0.05% Tween 80 to exponential phase (ODeoonm 0.4-0.6). The culture was diluted in RPMI-1640 media supplemented with 10% FBS to a cell density of ~2 x 106cells / ml (OD600nm = 0.05). The media in the wells containing adherent macrophages was removed, and 1 ml of the diluted mycobacterial inoculum (10 bacteria: 1 THP1 cell) was added to all wells and incubated for 4 hours at 37°C, 5% CO2to promote phagocytosis. The wells were then washed once with warmed RPMI-1640 media supplemented with 10% FBS media, and duplicate wells were treated with 7 different formulations (S1 -S7 ; 25, 108, 70, 124, 107, 180, 230 μg / ml respectively) as well as Vitamin D3 (VITD3, 100 μg / ml). Rifampicin (RIF) at 100X MIC (0,05 μg / ml) was included as the control together with untreated infected macrophages. Survival was monitored by enumerating colony- forming units (CFU / ml) after 5 days of incubation. Ten microliters of 10% SDS was immediately added to wells containing untreated infected macrophages to lyse the macrophages, serving as time zero. After 5 days, the treated and untreated macrophages were lysed with 10% SDS and appropriate dilutions of the supernatant was spread in duplicate on Middlebrook 7H11 solid media (Difco) supplemented with 10% OADC and 0,2% glycerol. The plates were incubated at 37°C for 4 weeks before enumeration of mycobacterial growth and analysis of CFU / ml. Two biological replicate experiments were conducted. 2.14. Statistical analysis

[0085] Statistical analysis was conducted using GraphPad Prism 10 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance (p) was determined using unpaired multiple t-tests with the Holm-Sidak correction method, considering p < 0.05 statistically significant. Each data set was analyzed individually without assuming a consistent standard deviation. Data is presented as mean values of biological samples ± SD.

[0086] 3. Results and Discussion

[0087] 3.1. Particle size distribution, zeta potential, PDI, stability, morphology, and drug entrapment efficiency

[0088] The average hydrodynamic particle size, zeta potential, PDI and drug entrapment efficiency for the formulated nanoparticles were measured and summarised in Table 1 and Fig. 1-2. The nanoparticles displayed an average hydrodynamic particle size within the range of 122.63 ± 5.11 nm to 182.90 ± 4.70 nm and a positive average zeta potential ranging from +19.5 ± 0.21 to +26.3 ± 0.45 mV, with narrow size distribution for all formulation (Pdl < 0.30) as shown in Table 1. Glucan functionalisation of the CS-PCL-VITD3 / RIF nanoparticles had minor changes to the average size, ZP and PDI as they remained in the same range as non-functionalized CS-PCL-VITD3 / RIF nanoparticles as seen in Fig. 1-2 and Table 1. The generated average particle size distribution, ZP, for the formulated nanoparticles showed the formulations' uniformity in size and zeta potential, supported by SEM results as displayed in Fig. 3. The formulated nanoparticles were demonstrated to have an average particle size ranging from 122.63 ± 5.11 nm to 182.90 ± 4.70 nm for optimal delivery of VITD3 / RIF payload to the macrophages.

[0089] Table 1: DLS properties of CS-PCL unloaded, loaded, functionalised and non- functionalised nanoparticles, and the drug loading capacity and entrapment efficiency.

[0090] Formulation Particle size ZP (mV) PDI (a.u) % LC % EE

[0091] (nm) .60 ± 2.51 +20.5 ± 0.40 0.28 ± 0.010

[0092] CS-PCL-VITD3 182.13 ± 4.70 +26.3 ± 0.45 0.24 ± 0.004 2.98 ± 0.35 62 ± 0.97

[0093] GLU-CS-PCL-VITD3 182.90 ± 1.15 +24.8 ± 0.73 0.15 ± 0.006 2.60 ± 0.22 54 ± 0.88

[0094] CS-PCL-RIF 156.13 ± 1.12 +20.6 ± 1.05 0.14 ± 0.027 4.60 ± 0.64 96 ± 1.03

[0095] GLU-CS-PCL-RIF 122.63 ± 5.11 +19.5 ± 0.21 0.27 ± 0.009 3.00 ± 1.01 62 ± 1.44

[0096] Another critical factor which influences the cellular uptake of nanoparticles is the surface charge. The formulated nanoparticles displayed positive average surface charge, as shown in Fig. 1 -2 and Table 1 , due to cationic surface coating by chitosan, thereby to enhance internalisation by cells, particularly macrophages via micropinocytosis and macrophage-particulate electrostatic interaction. In addition, the average surface charge (Fig. 1-2) and Pdl, as displayed in Table 1 of the formulated nanoparticles, indicated the stability and homogeneity of the nanoparticles in terms of aggregation, flocculation, sedimentation, coagulation and creaming as charged nanoparticles are kept apart by robust electrostatic repulsion between them

[0097] In addition to size, the shape of the nanoparticles also plays a pivotal role in the uptake pathway and trafficking of nanoparticles (Foroozandeh and Aziz, 2018). Therefore, surface electron microscopy (SEM) and Transmission Electron Microscope (TEM) were employed to evaluate the formulated nanoparticles' surface morphology and shape. The SEM and TEM images in Fig. 3 revealed that the prepared nanoparticles had spherical, smooth, uniform shape with intact structures. Fig. 3c and Fig. 3f of β- 1 ,3-Glucan functionalised nanoparticles presented a smooth, consistent spherical surface morphology not different from the non-functionalized nanoparticles in Fig. 3a and Fig. 3d. The morphology profile showed slight aggregation of the nanoparticles after the entrapment of the VITD3 and RIF (Fig. 3b). The spherical shape of the formulated nanoparticles enhances their internalisation by macrophages. Furthermore, the particle size distribution for all the nanoparticles was within the nanoscale, which correlated with the data obtained from the DLS as shown in Table 1.

[0098] Determination of VITD3 / RIF %LC and %EE of the synthesised nanoparticles was undertaken, as shown in Table 1. In the study, CS-PCL NPs exhibited a high % encapsulation efficiency of 96% for RIF. However, when these nanoparticles were modified with β-1 ,3-glucan, the encapsulation efficiency decreased to 62%. Similarly, with encapsulating VITD3, the initial encapsulation efficiency was 62%, which decreased to 54% after functionalisation with β-1 ,3-glucan. This reduction in encapsulation efficiency may be attributed to potential drug leaching that occurred during the incubation period for functionalisation. However, the observed encapsulation showed suitability for the encapsulation of VITD3 and RIF as the functionalisation aims to enhance the effectiveness of drug delivery at lower doses by improving target specificity and reducing any potential toxicity associated with high drug dosage. Furthermore, the degree of β-1 ,3-glucan functionalisation as a function of time was investigated. Results showed that the highest degree of functionalisation (41 .8%) was achieved at 24hrs of stirring time while stirring further than 24hrs had no significant effect on the degree of functionalisation (results not shown).

[0099] 3.2. characterisation of the

[0100] Fourier Transform Infrared Spectroscopy (FTIR) is used to study and characterise the nature of functional groups occurring at molecular surfaces. FTIR analysis was carried out for all excipient materials, non-functionalised and functionalised nanoparticles, and presented in Fig. 4-5. The pure PVA main peaks were assigned as follows, with the main prominent absorption peaks at 3297 cm-1(-OH stretching), 2910 cm-1(-CH stretching), 1731 cm-1(C=O stretching and -CH bending) and 1028 cm-1(-C-O- stretching) corresponding to literature (Abureesh M.gL.JQlS, Sgfl et et a L , 2021 ) . In the FTIR spectrum of CS, the following prominent peaks were also assigned; 3297 cm-1(-OH and -NH symmetric vibration), 2878 cm-1(-CH stretching), 1615-1515 cm-1(Amide I and Amide II stretching), 1424-1326 (-CH stretching), 1059-1027 cm-1(-C- O-stretching) and 895 cm-1(saccharine structure) as previously reported in the literature (Abureesh et al., 2016, Fernandes Queiroz et al., 2015, Suflet et 2021 ). The PCL prominent peaks were also characterised as follows: 2944 cm-1and 2863 cm-1(Asymmetric and symmetric -CH stretching), 1721 cm-1(C=O stretch), 1237 cm-1(asymmetric COC stretch) and 1161 cm-1(COC symmetric stretch and OC-O stretching) (Gorodzha 2015). The resulting non-functionalised nanoparticle (CS- PCL) consisted of combining all the starting materials with each component dispersed throughout the matrix without any chemical transformation, as seen in Fig. 4a. The characteristic peaks of PVA and CS were apparent on the resulting CS-PCL nanoparticles. The broadband at 3194 cm-1corresponded to the stretching vibrations of hydrogen-bonded -OH and -NH, which slightly shifted to lower wavenumbers while the -CH stretching peak at 2935 cm-1was retained (Fig. 4a). Furthermore, the fingerprint region of PCL in the ranger of 1161 -1000 cm-1was also present on the CS- PCL spectrum showing the presence of PCL and evidence of composition formation.

[0101] When VITD3 / RIF was incorporated within the non-functionalised CS-PCL nanoparticles, no bands or absorption peaks of either VITD3 / RIF were detected, signifying encapsulation of VITD3 / RIF within CS-PCL nanoparticles in the PCL matrix via hydrophobic interaction without the formation of new bonds as seen in Fig. 4b (El Yousfi et al., 2023). Upon surface functionalisation of the CP-PCL NPs with β-1 ,3- glucan, there were significant distinctions observed in the spectra of the functionalised Glu-CS-PCL nanoparticles, as highlighted in Fig. 5. The absorption spectrum of non- functionalised CS-PCL displayed an initial band at 1727 cm-1, which was notably intensified in functionalised Glu-CS-PCL. This enhancement can be attributed to the chemisorption and physisorption of β-1 ,3-glucan, resulting from interactions with hydroxyl groups and the C=O stretching vibration of the ester group (Trombino et al., 2022); additionally, the presence of β-1 ,3-glucan was evident in the functionalised Glu- CS-PCL nanoparticles spectrum within the spectral range of 1500-950 cm-1, as a new broader peak formed as highlighted in Fig. 5. This spectrum shows the successful surface functionalisation of the nanoparticles with β-1 ,3-glucan.

[0102] 3.3. X-ray powder diffraction analysis of the formulated nanoparticles and nanocomposite

[0103] The degree of crystallinity of the host polymer matrix is an essential parameter in the drug release study profile, with a higher drug release rate observed in polymers with a low degree of crystallinity, whilst delayed sustained drug release being observed in polymers with a higher degree of crystallinity (Jeong et al., 2003, Karavelidis et al., 2011). The crystallinity degree of formulated nanoparticle and all the excipients' materials using XRD technique was carried out and presented in Fig.8. The XRD pattern of pure chitosan, which is relatively amorphous, exhibits broad peaks at 29 ~ 10° and 20°, as depicted in the pattern obtained for pristine CS in Fig. 6a. Similarly, PCL, being semi-crystalline, displays two major peaks at 20 ~ 21° and 23°, corresponding to the pattern obtained for pristine PCL in Fig. 6a. Furthermore, the strong semi-crystalline diffraction peaks of PVA at 20 ~ 19° and 20° were also observed in the same region, consistent with prior literature reports respectively (Siafaka et al., 2016, Suganthi et al., 2020, Castilla-Cortazar et al., 2019). Formulated non-functionalised nanoparticle (CS-PCL) depicted high major crystalline diffraction peak at 9.6°, which might be attributed to polymer hybridisation to form crystalline nanoparticles (Abureesh st al., 2016, Jana et al., 2014).

[0104] The peak intensity of PCL (-18.8 - 23.36°) was significantly lowered, as observed in Fig. 6a-b. It is possible that the coordinate property of PCL polymer molecules was changed due to the presence or being incorporated within the CS / PVA shell, leading to the reduction of PCL peak intensities (Abureesh et al,. 2016, Jana et al., 2014). This phenomenon may signify good miscibility of component materials in the chitosan / PCL blend (Jana i et aL, ..2014). Two semi-crystalline peaks were further observed for CS- PCL from 18.8 - 23.36° and 23.5 - 25.34°. Other crystalline peaks with low intensities were further observed at 35.65° and 40.36 - 45°, confirming the crystallinity of the formulated nanoparticles. The crystallinity of non-functionalised CS-PCL observed is expected to contribute to sustained drug release.

[0105] The XRD pattern of pure VITD3 / RIF corresponded to the previously reported strong crystalline diffraction peaks, as displayed in Fig. 6a (Sharma s.et.aj., 2021 , ypra et al., 2018). After encapsulating the VITD3 / RIF, the diffraction patterns of nanoparticles loaded with either VITD3 or RIF resembled those of unloaded nanoparticles, as shown in Fig. 6b. These patterns did not exhibit any peaks related to the drug crystals. This observation indicates that the drug / bioactive was either in an amorphous state within the polymer matrix or dispersed within the amorphous region of the polymer matrix rather than being present in the crystalline region and further confirming successful drug incorporation within the nanoparticles (Jeong et al,, 2003, Bodmeier and McGinity, 1987). Following the surface functionalisation of CS-PCL nanoparticles with β-1 ,3-glucan, a noticeable alteration in the diffraction pattern of CS-PCL occurred. This change was attributed to the presence of β-1 ,3-glucan and manifested as two distinct crystalline peaks at approximately 21.5° and 23.5°, as illustrated in Fig. 6c. This observation confirms the presence of β-1 ,3-glucan in the modified nanoparticles. 3.4. Evaluation of the thermal behaviour of the optimised samples using Differential Scanning Calorimetry (DCS) and storage stability

[0106] Differential Scanning Calorimetry (DSC) was employed to assess the thermal characteristics, stability, and drug encapsulation behaviour of the formulated nanoparticles by analysing changes in thermal phase transitions resulting from controlled temperature changes driven by heat flow Thermal transitions for all excipient materials, as previously reported in the literature, were recorded and presented in Fig. 7, along with those of formulated melting peaks in non-functionalised CS-PCL nanoparticles at an approximate temperature of 166.31 °C was observed, a temperature higher than that of the main starting polymers, namely CS and PCL, as evidenced in Fig. 7a. This newly identified peak can be attributed to the interaction between the polymers when they are blended, indicating a sufficient degree of miscibility and interaction, resulting in a single-phase transition peak, a phenomenon in line with previous research findings (She et al., 2007). Upon loading the nanoparticles with VITD3 / RIF, it was observed that the endothermic melting peaks of the CS-PCL nanoparticles remained unaltered. At the same time, those corresponding to native VITD3 / RIF were absent, as illustrated in Fig. 7b. This observation implies that the encapsulation of VITD3 / RIF did not exert any discernible influence on the stability or thermal behaviour of the nanoparticles. It suggests that the drug molecules remained isolated from each other or were effectively dispersed within the nanoparticle matrix, thereby preventing them from undergoing bulk transition phases This assertion supports the data presented in Fig. 6 (XRD) and Fig. 4 (FTIR), further corroborating the notion of drug isolation and dispersion within the nanoparticle matrix.

[0107] Upon functionalising CS-PCL nanoparticles with β-1 ,3-glucan, a discernible enhancement in the PCL peak at 53.42°C was observed, a phenomenon consistent with the findings from FTIR analysis as depicted in Fig. 5. This enhancement can be attributed to the physisorption of β-1 ,3-glucan onto the nanoparticle surface, arising from interactions with hydroxyl groups and the C=O stretching vibration of the ester group This observation underscores the role of β-1 ,3-glucan in influencing the thermal properties of the CS-PCL nanoparticles, further elucidating the functionalisation process's impact on their thermal behaviour. Furthermore, the Glu-CS-PCL NPs thermogram had broader endothermic melting peaks at 94 and 226°C due to the interaction between β-1 ,3-glucan and CS-PCL NPs. The formulated nanoparticles, whether they were functionalised or not, demonstrated favourable thermal stability characteristics that are well-suited for polymeric nanoparticles and align with their intended applications, as evident from their consistently elevated melting points falling within the temperature range of 100-250°C (Mansfield and Banash. 2023, Mansour, 2013) This heightened thermal stability signifies that these nanoparticles possess the requisite resilience to withstand thermal conditions relevant to their intended purpose, bolstering their suitability for various applications in which thermal stability is a critical factor (Das A et a]., 2020, Mansour, 2013).

[0108] Nanoparticle stability encompasses multiple factors, including particle size, surface charge, and PDI, where stability is indicated by the minimal changes in particle size, zeta potential, and PDI over time, signifying resistance to aggregation or agglomeration and ultimately better stability, with a higher zeta potential contributing to stronger electrostatic repulsion between particles for enhanced stability, and a lower PDI denoting a more uniform size distribution, further reinforcing overall stability (Phan and Haes, 2019, Jain et al., 2018). The storage stability of the formulated nanoparticles was carried out using DLS after 3 months by measuring any changes in particle size, zeta potentials and PDI, as presented in Table 2. Particle size for all the formulations had an increase in range 10-25 nm, with only Glu-CS-PCL-RIF having a 48 nm increase, whilst the zeta potential for all formulations exhibited slight changes, including PDI. The observed increase in particle size within the range of 20-50 nm across all formulations suggests a notable but relatively modest alteration in the size of nanoparticles over three months of no more than 50 nm, which can be considered minor changes given that the nanoparticles are still within the acceptable range to be phagocytised by macrophage through phagocytosis or endocytosis mechanism (Lazzad,,,et..al;.L..2O12, .Kjjo,,, and, .Park,...2021.). This increase in particle size can be attributed to various factors, including potential aggregation or minor structural adjustments. The slight changes in zeta potential, as well as the persistence of consistent PDI values across all formulations, are indicative of the nanoparticles' ability to maintain their electrostatic stability and size distribution over the observed time frame, which thus effectively resist agglomeration or aggregation Hernandez-Gjgttonini.eta l,.2020)- The sustained stability of these nanoparticles over three months is a promising indication of their potential utility as drug delivery systems. Such stability is vital to ensure the reliability and effectiveness of nanoparticle-based formulations, as it suggests that the nanoparticles can retain their desired characteristics and functionality over extended periods.

[0109] Table 2: Stability profile of formulated nanoparticles as a function of time using DLS at room storage temperature

[0110] 3.5. In vitro release of VITD3 and RIF in formulated nanoparticles

[0111] The rate and quantity of drug release significantly impact dosing frequency. Extended- release formulations reduce the need for frequent administration, which is crucial for ensuring patient compliance with infectious diseases like tuberculosis (Sumaila et al., 2022). This study examined in vitro release profiles of encapsulated VITD3 and RIF from nanoparticles. These nanoparticles were either functionalised with β-1 ,3-glucan or non-functionalized. The experiments were conducted under various pH conditions (4.6, 6.8, and 7.4) using appropriate release media solutions, and the results are presented in Fig. 8 (for VITD3) and Fig. 9 (for RIF). The analysis of drug release kinetics, depicted in both Fig. 8 and Fig. 9, revealed a similar pattern between functionalised and non-functionalized nanoparticles.

[0112] Specifically, for VITD3, an initial burst release was observed at the 1 -hour mark, accounting for approximately 20%, 26%, and 30% of the total release at pH 7.4, 6.8, and 4.6, respectively, in both functionalised and non-functionalized nanoparticles (Fig. 8). Subsequently, between 4 to 48 hours, a sustained release phase occurred, as indicated by the plateau in the release curve. This phase resulted in cumulative releases of approximately 46%, 55%, and 78% at pH 7.4, 6.8, and 4.6, respectively, at the 6-hour mark. Notably, the highest drug release, reaching approximately 86%, was observed at pH 4.6, compared to 37% and 85% at pH 7.4 and 6.8 after 48 hours. Similarly, for RIF, an initial burst release of 11 %, 18%, and 14% at the 1 -hour mark was observed at pH 7.4, 6.8, and 4.6, respectively, for both functionalised and non- functionalized nanoparticles (Fig. 9). This initial release was followed by a sustained release phase, characterised by a plateau similar to what was observed for VITD3 in Fig. 8. RIF exhibited its maximum cumulative release at pH 4.6, with approximately 96% release, followed by pH 6.8 and 7.4, with 85% and 37% release, respectively, after 48 hours of drug release.

[0113] This observed release behaviour in Fig. 8 and Fig. 9 can be attributed to the interplay of factors such as pH-dependent nanoparticle degradation and the nanoparticle's structural properties The initial burst release observed at the 1 - hour mark may be attributed to the rapid dissolution of the drug molecules that are readily accessible on the nanoparticle surface (Maria et al., 2017). This phenomenon is a common occurrence in nanoparticle-based drug delivery systems; following the initial burst release, the drug encapsulated within the nanoparticles is subsequently released at a slower rate, often resulting in a biphasic release profile characterised by an initial rapid release phase followed by a more controlled and gradual release phase as observed in Fig. 8 and Fig. 9 (Sung et al. , 2009, Maria et al , 2017). Subsequently, as the release medium's pH influences the degradation of nanoparticles, variations in pH conditions can result in differential release rates Cha st al 2022). The elevated release rate of both VITD3 and RIF observed at pH 4.6 can be attributed to enhanced degradation of the nanoparticles in acidic environments. The plateau in drug release after reaching 86% (VITD3) and 96% (RIF) indicates that a significant portion of the drug payload has been released, and the system has reached equilibrium.

[0114] These results explain the drug release characteristics of the nanoparticles under varying pH conditions, offering valuable insights into their potential applications in drug delivery. Existing literature has shown that the pH within macrophages infected by Mtb rapidly shifts from a neutral range to approximately pH 4.5-5 during phagolysosome acidification, which facilitates bacterial degradation, and Mtb has been found to survive within these macrophages by inhibiting the biogenesis and acidification of the phagolysosome, thereby stabilising the phagosome pH between 6.2-6.5, which enables Mtb to persist and progress within the intracellular environment 20.17, As a result, the fabricated nanoparticles are strategically positioned to enhance the delivery of drug payloads to macrophages infected by Mtb. The nanoparticles have demonstrated a high drug release rate at pH levels of 4.6 and 6.8. These pH levels are critical for the survival and degradation of Mtb within the macrophages, indicating that the nanoparticles are effectively tuned to the intracellular environment of the infected macrophages which is expected to improve treatment outcomes by ensuring that drugs are released where they are most needed and in a manner that aligns with the survival mechanisms of Mtb.

[0115] 3.6. In vitro cytotoxicity assay of formulated nanoparticles

[0116] The effect of the formulated nanoparticles and pristine bioactive substance on the cellular metabolic activity of RAW 264.7 macrophage cell lines and their cytotoxicity was evaluated over 48 hours using a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation Kit I. This assessment provides valuable insights into the cytocompatibility of the synthesised nanoparticles, with cell viability percentages below 40% indicating potent cytotoxicity, percentages ranging from 40%- 60% suggesting moderate cytotoxicity, percentages within the range of 60%-80% indicating weak cytotoxicity, and percentages exceeding 80% being indicative of a non-toxic response (Iso, 2009, Lopez-Garcia et al., 2014). As shown in Fig. 10, the treatment with non-functionalised nanoparticle (CS-PCL) formulation and RIF had no significant effect on the cellular metabolic activity of the RAW 264.6 cell line when compared to control having cell viability of -100% at all concentration ranges showing cytocompatibility of formulated nanoparticles with RAW 264.6 macrophages cell line. When cells were treated with VITD3, potent cell cytotoxicity was observed at VITD3's highest concentrations (300 and 150μg / mL) with percentage cell viability of 2% and 38%, respectively, as depicted in Fig.12. In contrast, at moderate to low concentrations (75 and 38 μg / mL) there was no observed cell cytotoxicity as the percentage cell viability was above 100% respectively. When cells were treated with non- functionalised CS-PCL encapsulating VITD3 (CS-PCL-VITD3), there was no observed cell cytotoxicity at all concentration ranges (300-38μg / mL) as percentage cell viability was above 80%, signifying non-toxic effect and cytocompatibility of non-functionalised CS-PCL-VITD3 nanoparticles with RAW 264.6 macrophages cell line. Similarly, with RIF encapsulated non-functionalised nanoparticles (CS-PCL-RIF), there was no cell toxicity observed, as shown in Fig. 10. These experimental results revealed that encapsulating VITD3 into non-functionalised CS-PCL nanoparticles could help reduce the in vitro cytotoxicity of VITD3 at higher concentrations as observed.

[0117] A similar experimental investigation was conducted to assess the cytotoxicity and cytocompatibility of the nanoparticles formulated in the previous study after functionalisation with β-1 ,3-glucan. The concentrations ranged from 6.25 μg / mL to 25 μg / mL. As illustrated in Fig. 13, results demonstrated that β-1 ,3-glucan exhibited no to weak toxic effects on RAW cells, with a concentration-dependent response. Upon functionalising the nanoparticles with β-1 ,3-glucan, no observable toxic effects on the cells were detected. The percentage of cell viability at all concentrations exceeded 80%, as depicted in Fig. 13. Consequently, it can be inferred that β-1 ,3-glucan can be safely employed to actively target macrophage cells actively, demonstrating its non- toxic attributes and compatibility with RAW macrophages. Therefore, the formulated nanoparticles are expected to provide an effective delivery system capable of safely encapsulating high concentrations of VITD3 and RIF. This system can facilitate the sustained release of bioactive contents over an extended duration while maintaining therapeutic concentrations, thereby reducing dosing frequency and enhancing overall therapeutic efficacy. 3.7. Intracellular ROS Quantification

[0118] The production of reactive oxygen species (ROS) plays a crucial role in the immune system’s response to pathogens. As illustrated in Figure 11 , there were no significant differences in ROS generation between free RIF (71.01 ± 2.66 RFU), CS-PCL nanoparticles (73.88 ± 1.95 RFU), and CS-PCL-RIF (85.51 ± 9.06 RFU). However, Glu-CS-PCL-RIF treatment significantly increased ROS levels (131.74 ± 9.06 RFU) by approximately 1.5 and 1.8-fold compared to CS-PCL-RIF and free RIF (p < 0.001). Similarly, VITD3-treated cells exhibited higher ROS production (168.57 ± 2.66 RFU), significantly surpassing both CS-PCL (72.88 ± 1.32 RFU) and CS-PCL-VITD3 (82.71 ± 6.25 RFU) treatments by 2.3 and 2.0-fold, respectively (p < 0.001). Notably, Glu-CS- PCL-VITD3 nanoparticles also exhibited enhanced ROS production (138.47 ± 5.91 RFU), approximately 1.7-fold higher than CS-PCL-VITD3 (p < 0.001).

[0119] The observed increase in ROS levels following β-1 ,3-glucan functionalization can be attributed to the activation of dectin-1 receptors on macrophages. These receptors initiate signalling pathways that trigger ROS production, contributing to bacterial killing, macrophage polarization toward the pro-inflammatory M1 phenotype, and the induction of autophagy. OS-mediated pathways are critical for the macrophages' bactericidal activity and further amplify immune responses by upregulating inflammatory mediators such as TNF-α. The difference in ROS generation between pristine VITD3 and encapsulated formulations is consistent with the higher concentration of free VITD3 used (36 μg / mL versus 4 μg / mL in nanoparticles). VITD3 enhances ROS production by inducing antimicrobial peptides and oxidative bursts. Together, these results underscore the synergistic effects of β-1 ,3-glucan functionalization and VITD3 encapsulation on enhancing ROS levels and immune activation.

[0120] 3.8. Pro- and Anti-

[0121] TNF-a Quantification

[0122] CS-PCL nanoparticles significantly increased TNF-α production (1049.75 ± 15.91 μg / mL) compared to control (802.25 ± 12.37 μg / mL, p < 0.01). Functionalization with β-1 ,3-glucan further enhanced TNF-α secretion, with Glu-CS-PCL-VITD3 (1377.88 ± 32.70 μg / mL) inducing a 1.8-fold increase compared to control and a 1.2-fold increase compared to CS-PCL-VITD3 (1102.25 ± 58.33 μg / mL). A similar trend was observed for RIF nanoparticles, with Glu-CS-PCL-RIF treatments showing significantly higher TNF-α levels than non-functionalized nanoparticles (p < 0.01).

[0123] The enhanced TNF-α production can be linked to the ROS-mediated activation of transcription factors, such as NF-KB, which are crucial for pro-inflammatory cytokine expression. Additionally, β-1 ,3-glucan functionalization promotes macrophage activation demonstrating its immunostimulatory effects. High TNF-α levels are indicative of a robust pro-inflammatory response, which supports macrophage- mediated Mtb clearance through mechanisms such as phagosome maturation and autophagy.

[0124] Anti-Inflammatory Cytokines: IL-4 and IL-10

[0125] Both IL-4 and IL-10 levels were negligible or undetectable across all treatments. IL-4, a hallmark cytokine of M2 macrophages, is typically associated with anti-inflammatory responses. The low or negligible levels of IL-4 suggest that the formulations predominantly promote an M1 pro-inflammatory macrophage phenotype. Similarly, IL- 10, another anti-inflammatory cytokine, was non-quantifiable, further emphasizing the pro-inflammatory skew of the nanoparticle treatments. The lack of anti-inflammatory cytokine production aligns with the study's goal of enhancing host-directed immune responses against Mtb, where pro-inflammatory mediators play a dominant role. This polarization supports a macrophage phenotype conducive to pathogen clearance while minimizing immune suppression.

[0126] 3.9. Morphological Analysis of RAW 264.7 Macrophages: Untreated vs. Treated

[0127] Morphological analysis of RAW 264.7 macrophages revealed distinct differences between the untreated and treated groups. The untreated cells were small, round, and smooth, indicative of the M0 phenotype, representing a baseline, non-activated state. In contrast, the treated cells exhibited larger, irregular shapes, with elongated or spread-out morphology and more granular cytoplasmic extensions. These features are characteristic of the M1 phenotype, suggesting pro-inflammatory activation. Additionally, some treated cells displayed oval shapes, indicating partial differentiation towards the M2 phenotype or undifferentiated state. The elevated levels of ROS and TNF-α (Figure 11), markers of M1 activation, further support the conclusion that the nanoparticles predominantly promote an M1 polarization, enhancing the pro- inflammatory immune response.

[0128] 3.10. MIC Evaluation of Formulated Nanoparticles Against H37Rv and MDR11

[0129] Strains

[0130] The minimal inhibitory concentrations (MICs) of the nanoparticle formulations were evaluated to assess their antimicrobial efficacy against drug-susceptible Mtb (H37Rv) and multidrug-resistant Mtb (MDR11) strains (Table 3 and 4). This study specifically emphasized formulations containing RIF, given its known antibacterial activity, while acknowledging that VITD3 nanoparticles serve primarily an immunomodulatory role rather than exhibiting direct antimicrobial effects. Standard anti-tubercular drugs RIF, isoniazid, and streptomycin were included as controls for comparative purposes.

[0131] Nanoparticles without active drug encapsulation (S1 , CS-PCL) displayed an MIC of 25 μg / mL against both H37Rv and MDR11 strains, highlighting the inherent antibacterial properties of chitosan. Chitosan’s polycationic nature facilitates electrostatic interactions with bacterial cell membranes, disrupting membrane integrity and leading to cell death. While polycaprolactone lacks antimicrobial properties, its incorporation enhanced the mechanical stability and overall performance of the nanoparticles for biomedical applications.

[0132] Encapsulation of VITD3 into CS-PCL nanoparticles (S2) did not improve MIC values against the H37Rv strain compared to S1 , with an MIC of 25 μg / mL (Table 3). However, against the MDR11 strain, the MIC increased to 50 μg / mL, suggesting a modest reduction in efficacy. This observation aligns with the immunomodulatory role of VITD3, which enhances the host’s immune response rather than directly targeting bacterial cells.

[0133] Table 3. Minimum Inhibitory Concentration of Nanoparticle Formulations Against Susceptible Mycobacterium tuberculosis, H37Rv.

[0134] Nanoparticles encapsulating RIF (S3, CS-PCL-RIF) demonstrated significantly enhanced antibacterial activity (Table 3) against H37Rv; the MIC was 0.1 μg / mL on day 7 and 0.2 μg / mL on day 14, compared to the RIF standard (0.003 μg / mL on day 7 and 0.0125 μg / mL on day 14). Importantly, the calculated drug-equivalent RIF concentration within S3 was below the standard RIF MIC (0.0045 μg / mL on day 7; 0.0092 μg / mL on day 14). This finding shows the enhanced potency of the nanoparticle formulation, likely attributed to a synergistic interaction between RIF and chitosan. Functionalization with 1 ,3-β-glucan (S5, Glu-CS-PCL-RIF) further amplified bactericidal activity. Against H37Rv, S5 exhibited MICs of 0.05 μg / mL on day 7 and 0.1 μg / mL on day 14, demonstrating superior efficacy compared to S3 (Table 3). This enhancement can be attributed to 1 ,3-β-glucan’s ability to interact with the complex cell wall structure of Mtb, particularly its peptidoglycan-arabinogalactan-mycolic acid matrix. Functionalization likely facilitated closer nanoparticle-cell wall interactions, improving drug delivery efficiency and overall antimicrobial activity. Notably, the drug- equivalent MIC of S5 for RIF was 0.00149 μg / mL on day 7, significantly lower than the standard RIF MIC. This emphasizes the functionalization’s potential to reduce drug dosage while maintaining efficacy, strongly suggesting enhanced targeting efficiency attributable to the surface-functionalization strategy.

[0135] Combination therapy with S5 (Glu-CS-PCL-VITD3) and S4 (Glu-CS-PCL-RIF) in S7 demonstrated the greatest potency, achieving an MIC of 0.025 μg / mL against H37Rv (Table 3). This synergistic effect can be attributed to the combined functionalized nanoparticles, which enhance bacterial targeting and payload delivery. Interestingly, S7 exhibited notable efficacy against the MDR11 strain, achieving an MIC of 0.745 μg / mL of encapsulated RIF (four times lower than the RIF standard MIC >3.2 μg / mL) (Table 4). This result is particularly significant given MDR11’s resistance to RIF, suggesting that 1 ,3-β-glucan-functionalized nanoparticles can partially overcome drug resistance through enhanced interaction with the Mtb cell wall and improved delivery of sub-MIC drug concentrations.

[0136] The functionalized nanoparticles, particularly Glu-CS-PCL-RIF and the combination S7 demonstrate significant advancements in anti-tubercular therapy. These formulations not only enhance the efficacy of RIF against drug-resistant strains but also allow for a substantial reduction in drug dosage, potentially mitigating side effects and improving patient compliance.

[0137] Table 4. Minimum Inhibitory Concentration of Nanoparticle Formulations Against Drug-Resistant Mycobacterium tuberculosis MDR11 Strain.

[0138] 3.11. Intracellular Killing of Susceptible Mtb H37Rv in TH P-1 -Activated Macrophages The efficacy of the formulated nanoparticles was assessed using TH P-1 -activated macrophages infected with the Mtb H37Rv strain. These formulations encapsulated either RIF or VITD3, with each standardized to a drug concentration of 3.2 μg / mL (RIF / VITD3), as detailed in Table 5. Table 5: Concentration of nanoparticles and drug equivalent concentration used to treat H37Rv-infected THP-1 cells

[0139] The antibacterial response of the infected macrophages was compared between the nanoparticle formulations and a standard RIF treatment administered at 50 μg / mL, approximately tenfold higher than the MIC value determined earlier (Table 3 and 4).

[0140] 3.12. Efficacy of non-functionalized nanoparticles:

[0141] Among the nanoparticle formulations, CS-PCL (S1) exhibited CFU counts (2.15E+05 CFU / mL) comparable to the untreated control, highlighting its lack of intrinsic antibacterial activity against Mtb-infected macrophages. In contrast, CS-PCL-VITD3 (7.85E+04 CFU / mL) demonstrated significant bactericidal activity, showing that VITD3 can potentiate bactericidal mechanisms within macrophages. While CS-PCL-VITD3 was less potent than RIF (3.27E+03 CFU / mL), it achieved a significant (p < 0.005) reduction in bacterial load compared to both the untreated control and CS-PCL, further confirming that VITD3 plays a pivotal role in enhancing antibacterial activity.

[0142] 3.13. Mechanism of VITD3

[0143] Despite its higher MIC (50 μg / mL) relative to RIF (0.003 μg / mL), VITD3 displayed comparable bactericidal efficacy in infected macrophages, with CFU counts of 4.60E+03 and 3.27E+03 CFU / mL, respectively. This comparable activity is attributed to the ability of VITD3 to enhance macrophage-mediated bactericidal mechanisms, including the induction of antimicrobial peptides, RONS, autophagy, and cytokine- mediated immune modulation. VITD3 significantly increased ROS production and cytokine secretion in RAW 264.7 cells, suggesting a multifaceted enhancement of the innate immune response.

[0144] Addressing potential cytotoxicity, the concentration of VITD3 within CS-PCL-VITD3 nanoparticles (3.2 μg / mL) is well below levels shown to be non-toxic in this study, where macrophages treated with 145 μg / mL ofVITD3 exhibited >100% viability in MTT assays. Therefore, the observed bactericidal effects of CS-PCL-VITD3 can be attributed solely to the immunomodulatory and antibacterial properties of VITD3 rather than any cytotoxic effects.

[0145] 3.14. Efficacy of RIF-Containinq Nanoparticles

[0146] CS-PCL-RIF exhibited significant bactericidal activity compared to standard RIF treatment, with CFU counts of 4.05E+02 and 3.27E+03 CFU / mL, respectively (see Figure 12. This enhanced efficacy is particularly noteworthy given that the RIF concentration within CS-PCL-RIF was approximately 15 times lower than the standard RIF treatment. This improvement can be ascribed to the chitosan component of the nanoparticle formulation. Chitosan, a polycationic polymer, disrupts bacterial membranes through electrostatic interactions, enhancing the antimicrobial efficacy of encapsulated RIF as previously described. Additionally, the nanoparticle delivery system improves RIF bioavailability, protects against degradation, and ensures sustained drug release, collectively contributing to efficient bacterial killing. Similarly, Glu-CS-PCL-RIF nanoparticles reduced CFU counts to 1.81 E+03, further validating the advantages of functionalization in enhancing antibacterial activity. The synergy between the chitosan-mediated antibacterial effects and RIF’s potent bactericidal properties underscores the dual mechanism of action that optimizes drug delivery and efficacy.

[0147] 3.15. Functionalized nanoparticles 1 ,3-β-Glucan

[0148] Functionalized nanoparticles (Glu-CS-PCL-VITD3) outperformed their non- functionalized nanoparticles (CS-PCL-VITD3), achieving CFU counts of 1.08E+04 compared to 7.85E+04, respectively (Table 6). The enhanced efficacy of Glu-CS-PCL- VITD3 can be attributed to the 1 ,3-β-glucan functionalization, which targets Dectin-1 receptors on macrophages, facilitating nanoparticle uptake and intracellular delivery of VITD3. This functionalization exploits pathogen-mimicking properties to improve recognition, internalization, and subsequent antibacterial activity. Furthermore, 1 ,3-β- glucan functionalization promotes M1 macrophage polarization through increased ROS and pro-inflammatory cytokine production, augmenting the bactericidal response. Consistent with the MIC data, the bacterial killing assay further demonstrated that the functionalized nanoparticles achieved superior antimicrobial activity at lower concentrations compared to the non-functionalized system and standard drug. This enhanced potency supports the hypothesis that functionalization improves cellular interaction and delivery to the bacterial site, enabling effective killing at reduced bioactive levels.

[0149] Table 6: Intracellular Survival of M. tuberculosis H37Rv in THP-1 Macrophages like cells Treated with Various Nanoparticle Formulations

[0150] 3.16. Combined Formulation treatment

[0151] The combination of RIF- and VITD3-loaded nanoparticles (S6 and S7) demonstrated remarkable antibacterial activity, achieving CFU counts of 1.53E+03 (S6) and 1.00E+03 (S7), comparable to or exceeding standard RIF treatment (3.27E+03

[0152] CFU / mL). Notably, S7 (functionalized formulation) outperformed S6 (non- functionalized formulation), highlighting the synergistic benefits of 1 ,3-β-glucan functionalization and the dual action of RIF and VITD3. While RIF directly targets Mtb, VITD3 enhances autophagy and immune-mediated bacterial clearance, collectively providing a robust strategy for combating Mtb infections. The co-delivery of a single frontline anti-TB drug at reduced concentration with low-dose Vitamin D3offers a promising strategy for reducing cytotoxicity and overall treatment burden. 4. Conclusion

[0153] The polymeric macrophage-targeting nanoparticles exhibited excellent stability, homogeneity, and thermal resilience, making them suitable for drug delivery. Characterization of the nanoparticles showed strong physicochemical properties, optimal size for macrophage targeting, stability, cytotoxicity, and compatibility with macrophage cell lines.

[0154] The enhanced bactericidal activity of the polymeric macrophage targeting nanoparticles encapsulating RIF was attributed to the synergistic effects of each of the components present in the polymeric macrophage targeting nanoparticles of the invention together with RIF.

[0155] Functionalization with 1 ,3-β-glucan significantly improved nanoparticle uptake by macrophages and enhanced immune responses, as evidenced by increased ROS production and TNF-α levels, promoting M1 macrophage polarization. The nanoparticles exhibited potent antibacterial activity, with superior efficacy at lower drug concentrations compared to standard treatments, particularly against susceptible H37Rv and drug-resistant MDR11 Mtb strains.

[0156] The targeting function of the delivery system of the invention is derived from 1 ,3-3- glucan-functionalization. Mycobacterium tuberculosis contains specific molecules known as pathogen-associated molecular patterns (PAMPs), which include components of its cell wall such as lipopolysaccharides, mycolic acids, and glucans like arabinogalactan and peptidoglycan. The peptidoglycan layer is linked to arabinogalactan, which, in turn, interacts with mycolic acids to form the complex and unique cell wall structure of Mtb. The peptidoglycan-arabinogalactan complex can also interact with other glucans through glycosidic linkages, such as 1 ,3-β-glucan.

[0157] Expectedly, since β-glucan stimulates the immune system to target and fight infectious diseases by binding to cell-surface receptors, such as Dectin-1 , on immune cells like macrophages and neutrophils, the delivery mechanism of the present invention would be expected to provide β-glucan binding triggering intracellular signalling pathways that activate these immune cells, enhancing their ability to combat different bacterial, fungal, viral, and parasitic infections, β-glucan can also be used in trained immunity- based vaccines to provide broad, long-lasting protection against various infectious agents.

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Claims

CLAIMS1 . A polymeric macrophage-targeting nanoparticle delivery system comprising: an outer chitosan shell functionalized with 1 ,3-β-glucan and optionally at least one active compound; a hydrophobic polymer core comprising an immunomodulating agent and optionally at least one active compound; wherein where there is no active compound provided in the outer chitosan shell, there is an active compound provided in the hydrophobic polymer core and wherein where there is no active compound provided in the hydrophobic polymer core there is an active compound provided in the outer chitosan shell.

2. The polymeric macrophage-targeting nanoparticle delivery system according to claim 1 , wherein the hydrophobic polymer core is comprised or consists of a polymer selected from polylactide or polylactic acid (PLA), polyglycolide or polyglycolic acid (PGA), polycaprolactone, polydioxanone and a range of their copolymers. In particular, the hydrophobic polymer core is a polycaprolactone core.

3. The polymeric macrophage-targeting nanoparticle delivery system according to claim 1 or 2, wherein the immunomodulating agent is selected from cholecalciferol (VITD3), vitamin A, vitamin C, vitamin E, and β-carotene, dehydroascorbic acid, metformin, microelements including zinc, selenium, iron, omega-3 fatty acids, and live active probiotic bacteria.

4. The polymeric macrophage-targeting nanoparticle delivery system according to claim 3, wherein the immunomodulating agent is VITD3.

5. The polymeric macrophage-targeting nanoparticle delivery system according to any one of claims 1 to 4, having a size of between about 100 nm to about 200 nm, or from about 120 nm to about 190 nm, or any size range therebetween.

6. The polymeric macrophage-targeting nanoparticle delivery system according to any one of claims 1 to 5, which comprises more than one active compound.

7. The polymeric macrophage-targeting nanoparticle delivery system according to any one of claims 1 to 6, wherein at least one active compound is hydrophobic or hydrophilic.

8. The polymeric macrophage-targeting nanoparticle delivery system according to claim 7, wherein one active compound is hydrophobic and the other hydrophilic.

9. The polymeric macrophage-targeting nanoparticle delivery system according to claim 7, wherein the at least one active compound is one hydrophobic one active compound.

10. The polymeric macrophage-targeting nanoparticle delivery system according to any one of claims 1 to 9, wherein at least one active compound is an antibiotic, antifungal, antiviral, antimalarial, anti-TB drug, or antiretroviral.11 . The polymeric macrophage-targeting nanoparticle delivery system according to claim 10, wherein the active compound is Rifampicin (RIF).

12. The polymeric macrophage-targeting nanoparticle delivery system according to any one of claims 1 to 9, which is formulated for oral, transdermal, buccal, pulmonary, nose-to-brain, or intravenous drug delivery, or is comprised within a hydrogel or thermogel delivery system for prolonged and controlled release of the active compound to be delivered.

13. A pharmaceutical composition comprising a polymeric macrophage-targeting nanoparticle according to any one of claims 1 to 12 for macrophage-targeted delivery of at one active compound and optionally a pharmaceutically acceptable excipient.

14. A method for preparing the polymeric macrophage-targeting nanoparticle of the invention comprising the following steps: a) providing a hydrophobic polymer solution in a polar, aprotic solvent and further comprising an immunomodulatory agent and optionally an active compound as the organic phase;b) providing a solution comprising polyvinyl alcohol (PVA), chitosan oligosaccharide lactate, and optionally an active compound; c) adding, dropwise, the organic phase of a) to the solution of b) under stirring to yield a solution; d) sonicating the solution of c), followed by stirring to evaporate excess polar, aprotic solvent to form a solution of nanoparticles comprising an outer chitosan shell and optionally at least one active compound, and a hydrophobic core comprising the immunomodulatory agent and optionally at least one active compound, wherein where there is no active compound provided in the outer chitosan shell, there is an active compound provided in the hydrophobic polymer core and wherein where there is no active compound provided in the hydrophobic polymer core there is an active compound provided in the outer chitosan shell; e) collecting the solution of nanoparticles of d) for 1 ,3-β-glucan functionalisation; f) incubating the solution of nanoparticles with a 1 ,3-β-glucan solution thereby to attach the 1 ,3-β-glucan to the outer chitosan shell thereby to generate the polymeric macrophage-targeting nanoparticles; g) purifying the polymeric macrophage-targeting nanoparticles by centrifugation and washing; and i) optionally lyophilising the purified polymeric macrophage-targeting nanoparticles.

15. The method according to claim 14, wherein the hydrophobic polymer is selected from the group of polymers comprising or consisting of polylactide, polyglycolide, polycaprolactone, polydioxanone and any copolymers thereof.

16. The method according to claim 15, wherein the hydrophobic polymer is polycaprolactone.

17. The method according to any one of claims 14 to 16, wherein the polar, aprotic solvent is selected from the group comprising dicholoromethane (DCM), acetone, ethanol, or a mixture thereof.

18. The method according to claim 17, wherein the polar, aprotic solvent is DCM.

19. The method according to any one of claims 14 to 16, wherein the immunomodulating agent is selected from the group comprising cholecalciferol (VITD3), vitamin A, vitamin C, vitamin E, and β-carotene, dehydroascorbic acid, metformin, microelements including zinc, selenium, iron, omega-3 fatty acids, and live active probiotic bacteria.

20. The method according to claim 19, wherein the immunomodulating agent is VITD3.21 . The method according to any one of claims 14 to 16, wherein the at least one active compound is an antibiotic, antifungal, antiviral, antimalarial, anti-TB drug, or antiretroviral.

22. The method according to claim 21 , wherein the active compound is Rifampicin (RIF).

23. A method of administering a pharmaceutically active compound to a subject in need thereof, the method comprising a step of administering a polymeric macrophage- targeting nanoparticle as described in any one of claims 1 to 12 or a pharmaceutical composition as described in claim 13 to the subject.

24. A polymeric macrophage-targeting nanoparticle delivery system as described in any one of claims 1 to 12 or a pharmaceutical composition as described in claim 13 for use in a method of administration of a pharmaceutically active compound to subject in need thereof.

25. The polymeric macrophage-targeting nanoparticle delivery system or pharmaceutical composition according to claim 24, wherein the administration is selected from the group comprising oral, transdermal, buccal, pulmonary, nose-to-brain, or intravenous administration, or administration by a hydrogel or thermogel delivery system comprising the polymeric macrophage-targeting nanoparticle delivery system or pharmaceutical composition.

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