Hybrid Curdlan poly(γ-glutamic acid) nanoassembly for immune modulation in macrophage

Abstract

A nanoformulation composed of curdlan, a linear polysaccharide of 1,3-β-linked D-glucose units, hydrogen bonded to poly(γ-glutamic acid) (PGA), was developed to stimulate macrophage. Curdlan/PGA nanoparticles (C-NP) were formulated by physically blending curdlan (0.2 mg mL⁻¹ in 0.4 M NaOH) with PGA (0.8 mg mL⁻¹). Forster resonance energy transfer (FRET) analysis demonstrates a heterospecies interpolymer complex formed between curdlan and PGA. The ¹H-NMR spectra displayed significant peak broadening as well as downfield chemical shifts of the hydroxyl proton resonances of curdlan, indicating potential intermolecular hydrogen bonding interactions. In addition, the cross peaks in ¹H-¹H 2D-NOESY suggest intermolecular associations between the OH-2/OH-4 hydroxyl groups of curdlan and the carboxylic-/amide-groups of PGA via hydrogen bonding. Intracellular uptake of C-NP occurred over time in human monocyte-derived macrophage (MDM). Furthermore, C-NP nanoparticles dose-dependently increased gene expression for TNF-α, IL-6, and IL-8 at 24 hr in MDM. C-NP nanoparticles also stimulated the release of IL-lβ, MCP-1, TNF-α, IL-8, IL-12p70, IL-17, IL-18 and IL-23 from MDM. Overall, we are the first to demonstrate a simplistic nanoformulation formed by hydrogen bonding between curdlan and PGA that modulates cytokine gene expression and release of cytokines from MDM.

Graphical Abstract

We are the first to demonstrate a nanoformulation formed by hydrogen bonding between curdlan and PGA that modulates cytokine gene expression and release of cytokines from MDM

Background:

Curdlan (Beta-1,3-glucan), a non-pathogenic microbial polysaccharide, is a naturally occurring linear polysaccharide composed of 1,3-β-linked D-glucose units produced by Alcaligenes faecalis. Particulate curdlan binds to Dectin-1 expressed on immune cells such as dendritic cells, monocytes, macrophages, and B-cells, and stimulates immune cells to produce immune modulators such as cytokines ^{[1, 2]}. It potentially has an inhibitory effect against HIV infection ^{[3, 4]}, antibacterial therapy against tuberculosis (TB) ^{[5, 6]}, as well as blood anti-coagulant activity ^[7].

Previous studies have utilized curdlan as structural components of nanoparticles that modulate the immune system. Studies have conjugated curdlan oligo-saccharide units to the surface of polymeric/inorganic nanoparticles ^[8–10]. Conjugated curdlan to the surface of poly (D,L-lactic-co-glycolic acid) (PLGA) chitosan (-CS) nanoparticles stimulates cytokine production from macrophage ^{[5, 11, 12]}. In addition to surface bound curdlan, chemically modifying curdlan with small molecules induces self-assembling nanoparticles. For example, water-soluble carboxymethylated curdlan chemically modified with deoxycholic acid self-assembles through hydrophilic-hydrophobic interactions ^{[13, 14]}. Aminated curdlan electrostatically interacting with polynucleotides self-assembles into nanoparticles ^{[10, 15]}. Interestingly, chemical modification of curdlan with small molecules modifies its ability to stimulate cytokine production. As studies using aminated curdlan nanoparticles do not elicit cytokine production in macrophage, in fact, they were used to delivery siRNA directed against TNFα ^{[10, 15]}. It is well-established that particulate glucans are necessary to engage the dectin-1 receptor and activate macrophage ^[16]. Studies above demonstrate that chemical modification of curdlan with small molecules alters the size of curdlan and/or alters its binding capabilities to dectin-1 receptors.

We have previous published studies using curdlan as a targeting ligand to macrophage ^{[5, 6, 11]}, however, synthesis of these nanoparticles is a complex, multi-step synthesis process that is not feasible for scale up processes. Therefore, we sought to develop a nanoformulation of curdlan that had the following properties; 1) simple synthesis and 2) retains ability to stimulate macrophage. This was done by physically blending curdlan with poly(γ-glutamic acid) (PGA) to create a nanoparticle through hydrogen bonding. PGA is a promising bio-generated bio-degradable polymer that shares many functions with poly(acrylic acid) and its derivatives ^[17]. It can form interpolymer heterospecies complexes via multivalent hydrogen bonding in water, without further chemical modification, and its multitude of carboxylic acids act as moieties for further functional conjugation to chemotherapeutic drugs and diagnostic fluorophores for bioimaging ^[18]. Hydrogen bonding is a major driving force in self-assembly. Nucleic acids and proteins utilize hydrogen bonding for molecular recognition, and hyperstructure formation ^[19]. Multi-hydroxyl carbohydrates are implicated in cell-cell recognition, infection by pathogens, and many other aspects in the immune response via hydrogen bonding between hydroxyl groups ^[20]. Multivalent strong hydrogen bonding is attained in an adequately controlled or stereoselective fashion to overcome the interference by hydrogen bondable water molecules in the medium, and this property is used to generate stable nanoparticles in water ^{[21, 22]}. In specific, poly(carboxylic acid)-based proton donors and nonionic proton accepting polymers can spontaneously form heterospecies interpolymer complexes via multivalent hydrogen bonding in water ^[23].

We synthesized hybrid curdlan/PGA nanoparticles (C-NPs) by physically blending curdlan and PGA, which we demonstrate are capable of robust hydrogen bonding with each other, to explore the immune-modulator capability of artificial hydrogen bonded nanoassemblies in human monocyte-derived macrophage (MDM). To the best of our knowledge, this is the first study to evaluate a simple hydrophilic, biocompatible curdlan based nanoparticle formed with polyvalent hydrogen bonding with PGA, as an immune modulator of MDM.

Methods

Materials and Characterization:

Curdlan (Mw 1.1 × 10⁶ g mol⁻¹) was purchased from Sigma Aldrich. PGA (Mw 2.0 × 10 ⁵ – 5.0 × 10 ⁵ g mol⁻¹) was purchased from Wako Chemicals, Japan. All other chemicals and solvents were purchased from Sigma Aldrich and used without further purification. Transmission electron microscopic (TEM) images were recorded with a JEM-2010 microscope at an acceleration voltage of 200 kV. For the TEM sample preparation, a drop of diluted nanoparticle dispersion was dried on a 300 mesh copper grid supported with formvar film and negatively stained with 2% phosphotungstic acid solution. The size of dispersed nanoparticles in water was determined by dynamic light scattering (DLS) using a Brookhaven 90 Plus Particle Analyzer (Brookhaven Instruments, Holtsville, NY). Fluorescence spectra were acquired using a spectrophotometer (HORIBA Jobin Yvon) using a xenon lamp as the excitation source. ¹H NMR spectra were obtained from a Varian Inova Spectrometer operating at 500 MHz in a 5 mm tube using a PFG triple resonance probe. All spectra were obtained with standard pulse sequences provided by the vendor. Data presented were obtained using the standard 1D, Diffusion-Ordered Spectroscopy (BPPSTE-DOSY), and 2D-Nuclear Overhouse Effect Spectroscopy (2D-NOESY) experiments. All experiments were conducted in DMSO-d6 to monitoring hydroxy groups of curdlan. All data processing was performed using the MestreNova software version 12.0.3.

Preparation of hybrid curdlan/PGA nanoparticles (C-NPs):

5 mg mL⁻¹ solution of curdlan was prepared in 0.4 M NaOH solution at 55°C under vigorous stirring. The solution was syringe filtered with a 5 μm pore size. The 20 mg mL⁻¹ stock solution of PGA was prepared in 0.4 M NaOH solution. The 0.2 mL of the PGA solution was added into the aliquot of 0.2 mL of curdlan solution. The mixture was stirred for 30 min while maintaining at 55°C to make an optically homogeneous solution, followed by the addition of 4.8 mL of water during sonication. The mixture was cooled to room temperature resulting in an optically turbid suspension.

Conjugation of fluorescent dyes to curdlan and PGA:

Curdlan (50 mg) was labeled by direct conjugation with a tetramethylrhodamine B isothiocyanate (TRITC) (0.4 mg, 0.0004 mmol). PGA (50 mg) activated with N-(3-dimethylaminopropyl)-N’-ethylcarboiimide (EDC) (4 mg, 0.02 mmol) and N-hydroxysuccinimide (NHS) (2.5 mg, 0.02 nmol) was labeled with fluoresceinamine (0.1 mg, 0.0003 mmol). Each conjugation was stirred in DMSO for 12 h at room temperature in dark condition and dialyzed with a Spectra/Por membrane (MWCO: 7,000) against distilled water for 2 days.

Forster resonance energy transfer (FRET):

To confirm the C-NPs were heterospecies, composed of curdlan and PGA, Forster resonance energy transfer (FRET) was examined using TRITC-labeled curdlan and fluorescein (FITC)-labeled PGA. Because FRET is known to typically occur when the distance between an energy donor and its acceptor is in the range of 1 – 10 nm, we hypothesized that FRET between FITC as the excitation energy donor and the TRITC accepter would occur only when curdlan and PGA exist together in the resulting nanoparticle. The mixtures of FITC-labeled PGA + unlabeled PGA and TRITC-labeled curdlan + unlabeled curdlan were separately prepared to escape fluorescence quenching caused by conformational change of the conjugated polymers. Unlabeled polymers were added into labeled polymers until fluorescent intensity was maximumly constant. To prove the validity of this idea, the C-NP solution was prepared in the final concentration of 0.2 mg mL⁻¹ of curdlan and 0.8 mg mL⁻¹ of PGA, and diluted ten times more in water to remove the possibility of FRET due to interparticle-colloidal interactions.

In vitro analysis of curdlan nanoparticles:

Human peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Paque (GE Health Care, Piscataway, NJ) gradient centrifugation. CD14+ cells were isolated from PBMCs by direct positive isolation using Dynabeads CD14 (Thermo Fisher Scientific) according to the manufacturer’s instructions. CD14⁺ cells were cultured in complete medium [RPMI 1640, 10% fetal calf serum, 5% human AB serum, 10 mM HEPES, 1% Penicillin-Streptomycin, 10 ng mL⁻¹ macrophage colony-stimulating factor (Millipore, Billerica, MA)] for 7 days for differentiation into MDMs ^[24]. This study was approved by the University at Buffalo’s Institutional Review Board (IRB) committee, and the procedures followed are in accordance with institutional guidelines.

Cellular uptake:

MDM (1 × 10⁶ cells mL⁻¹) were incubated with 150 ng mL⁻¹ hybrid curdlan nanoparticles for 2, 4, 8, 12, 24 or 48 hr at 37°C. Cells were then fixed with 4% paraformaldehyde at 37°C for 10 min. Cells were subsequently incubated with the nuclear stain 4’,6-diamidino-2-phenylindole (DAPI). Macrophage were imaged using EVOS^® FL Cell Imaging System (Thermo Fisher Scientific) at 40x magnification. The light cube used to visualize DAPI nuclear staining had an excitation wavelength of 357/44 nm and emission of 447/60 nm. The light cube use to visualize TRITC labeled nanoparticles had an excitation of 531/40 nm and emission of 593/29 nm. Quantification of imaging was performed by densitometry using Image-J.

Toxicity:

MDM (10,000 cells/ml/well) were incubated for 24 hr with 150 ng mL⁻¹ of PGA solution, 150 ng mL⁻¹ curdlan solution, or 150 ng mL⁻¹ of curdlan nanoparticles. MDM were subsequently incubated with the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent for 3 hr (Thermo Fisher Scientific), followed by the addition of sodium dodecyl sulfate to lyse the cells and solubilize the colored crystals. Cell suspensions were read using an ELISA plate reader (Biotek) at a wavelength of 570 nm, quantifying formazan as an indicator of the number of viable cells.

Gene expression:

MDM were incubated with 18.75, 37.5, 75, 150, or 300 ng mL⁻¹ of the hybrid curdlan nanoparticles for 24 hr. Cells were then washed in PBS, and RNA was extracted by an acid guanidinium-thiocyanate-phenol-chloroform method using the Trizol reagent (Thermo Fisher Scientific). The amount of RNA was quantitated using a Nano-Drop ND-1000 spectrophotometer (Thermo Fisher Scientific). The extracted RNA was reverse transcribed to cDNA using a reverse transcriptase kit (Promega Inc, Madison, WI) by following the manufacturer’s instruction. The relative abundance of each mRNA species was quantitated by normalizing the expression levels of each mRNA to the endogenous β-actin levels. Q-PCR was done using specific primers to IL-8, TNF-α, IL-6, and IL-10. All primers were validated for efficiency and specificity by qPCR with melt curve analysis, and gel electrophoresis. Brilliant SYBR green Q-PCR master mix (Stratagene Inc., La Jolla, CA) was used. Q-PCR was performed in a reaction volume of 25 μL. Briefly, 12.5 μL of master mix, 2.5 μL of assay primers (10×), and 10 μL of template cDNA (100 ng) were added to each well. After a short centrifugation, the PCR plate was subjected to 35 cycles under the following conditions: (i) PCR activation at 95°C for 5 min, (ii) denaturation at 95°C for 5s, and (iii) annealing/extension at 60°C for 10s. All samples and controls were run in triplicate on a Stratagene MX3000P real-time PCR system. To provide a precise quantification of the initial target in each PCR reaction, the amplification plot was examined, and the data were calculated as described. Relative expression of mRNA species was calculated using the comparative threshold cycle number (C_T) method ^{[25, 26]}. Briefly, for each sample, a difference in C_T values (ΔC_T) was calculated for each mRNA by taking the mean C_T of duplicate tubes and subtracting the mean C_T of the duplicate tubes for the reference RNA (β-actin) measured on an aliquot from the same RT reaction. The ΔC_T for the treated sample was then subtracted from the ΔC_T for the untreated control sample to generate a ΔΔC_T. The mean of these ΔΔC_T measurements was then used to calculate the levels in the targeted cytoplasmic RNA relative to the reference gene, and normalized to the control as follows: relative levels or transcript accumulation index (TAI) = 2^−ΔΔC_T. This calculation assumes that all PCR reactions are working with 100% efficiency. All PCR efficiencies were found to be >95%; therefore, this assumption introduced minimal error into the calculations.

Cytokine production:

MDM were incubated with 150 ng mL⁻¹ of PGA solution, 150 ng mL⁻¹ curdlan solution or 150 ng mL⁻¹ of curdlan nanoparticles for 4 hrs. Supernatants from MDM were assayed using a bead-based immunoassay that uses the principles of sandwich ELISA to quantify soluble analytes using a flow cytometer (LEGENDplex Multi-Analyte Flow Assay Kit, Human Inflammation Panel 1, BioLegend). The concentrations of IL-lβ, TNF-α, MCP-1, IL-8, IL-10, IL-12p70, Il-17, IL-18 and IL-23 were determined as per manufacturer’s instruction. Data were collected on a Fortessa five laser flow cytometer (Becton Dickinson) and analyzed using BioLegend’s cloud-based LEGENDplex™ Data Analysis Software.

Statistical Analysis

Statistical analysis was performed using one-way ANOVA (Prism 8) followed by Tukey’s multiple comparisons test (post-hoc test).

Results:

Colloidal behavior of the heterospecies interpolymeric self-assembly in water:

The behavior of the aqueous self-assembly between curdlan and PGA was studied. PGA solution was added into to curdlan as followed: 1:8, 1:4, 1:2, 1:1, 1:0.1, 1:0.01, and 1:0 (wt/wt). The samples of 1:1, 1:0.1, and 1:0.01 were optically clearly precipitated and the samples of 1:2 and 1:1 were precipitated over time. Because colloidal stability over time is vital in biomedical application, the samples of 1:2 and 1:1 were excluded for further study as well. An optically stabilized solution of C-NP was obtained when PGA was added over 4-times (wt%) to the amount of curdlan, while the cooled curdlan only solution without PGA formed micron-sized aggregates (data not shown), which indicates that PGA helps control the resulting colloid size. The clear solution with 0.2 mg mL⁻¹ of curdlan and 0.8 mg mL⁻¹ of PGA, was examined by DLS, and the dried nanoparticles were analyzed by TEM. Nanoparticles were found to have an average size of 168.8 ± 15.9 nm (Figure. 1a). Using FRET, we observed that the fluorescence intensity of TRITC increased in a 10 folds diluted solution, when the FITC donor was excited at 480 nm (Figure. 1b). This indicates that heterospecies interpolymer complexes between curdlan and PGA formed in the colloidal nanoscopic domain. In addition, the change of zeta-potential of the nanoassembly showed a mixed phase on the surface of the nanoparticles, suggesting the coexistence of curdlan and PGA (Figure. 1c). The structure of the colloids was further examined with varying pH. As shown in Figure. 1d, the size of the colloid increases as pH decreases. This size swelling is attributable to the loosened hydrogen bonding by the proton interference under acidic condition. Additionally, at pH 7, the colloidal structure remained stable for 9 days (Figure. 1e). These results suggest the possibility of the nanoassemble as a sustainable drug delivery carrier, and targeting and release of the therapeutic at a low pH in the cellular compartment such as lysosome.

Figure 1.

(a) Size distribution of C-NPs in water and TEM image; (b) Forster resonance energy transfer (FRET) in C-NP (red) composed of TRITC-labeled curdlan and fluorescein-labeled PGA, compared with the fluorescence of TRITC-donor labeled curdlan (blue), and FITC-acceptor labeled PGA (black); (c) Zeta-potential measurement of PGA, curdlan, and C-NPs; (d) pH-dependent size difference of C-NP; and (e) colloidal stability of C-NP at pH 7.0.

Hydrogen bond mediated nanoassembly of curdlan and PGA:

The presence of hydrogen bonding between curdlan and PGA was investigated by analyzing the titration profiles of the hydroxyl proton resonances of curdlan (OH-2, OH-4, and OH-6) with increasing PGA concentration (Figure. S2). Significant broadening as well as downfield chemical shifts of the hydroxyl proton resonances of curdlan can be seen in NMR, with increasing PGA concentration, demonstrating potential intermolecular hydrogen bonding interactions even in DMSO which is a highly solubilizing solvent where the hydrogen bonds readily dissociated at low concentrations ^{[27, 28]}. The broadening of NMR peaks at higher PGA concentrations indicates the presence of aggregated larger species. Therefore, under the 1:2 ratio of curdlan: PGA, the system is well organized and ordered for NMR analysis. Hence this ratio was further investigated as described below.

To ascertain the nature of the interaction and possible complex formation between curdlan and PGA, we recorded the NMR Diffusion-Ordered Spectroscopy (DOSY) data for a 1:2 mixture (wt%) of curdlan and PGA in DMSO-d₆ at 25°C. The DOSY data are presented in Figure. S3. As is evident from the DOSY data (Figure. 3), under our experimental conditions, curdlan and PGA exist together as a single unit, indicated by a single diffusion constant for all ¹H resonances of both curdlan and PGA. It is not possible to ascertain the molecular organization of the complex from this data, but the DOSY data strongly suggests that they are organized together in a 1:2 ratio of curdlan and PGA. We then performed a ¹H-¹H 2D- Nuclear Overhauser Effect Spectroscopy (NOESY) experiment to ascertain molecular interactions between curdlan and PGA in the above postulated complex. The NOESY data detects protons that are closer than 5Å in space. Figure. 2. is an expanded region of NOESY spectrum, showing the region of interest: the region of contact between the OH-2 and OH-4 protons of curdlan with the alpha protons of PGA. The presence of the NOESY cross peak between these sets of protons indicates their proximity in space and correlates with possible intermolecular hydrogen bonding. More specifically, this interaction probably is due to hydrogen bonding between the OH-2 and OH-4 groups of curdlan with the carboxyl or amide moieties of PGA. More importantly, the NOESY spectrum revealed that OH-2 and OH-4 may be more relevant for complex formation due to the absence of space contact between OH-6 and PGA. Finally, to confirm the presence of the 1:2 curdlan: PGA complex stabilized by hydrogen bonds, we investigated the effect of temperature on the complex ^{[29, 30]}. As shown in Figure. S4, the peaks implicated in the intermolecular hydrogen bonding of the complex, show upfield shifts of the resonances as well as extreme line broadening for hydroxyls OH-2,4 and 6 of curdlan as a function of increasing temperature. This result correlates with the weak nature of the interaction, which is a hydrogen bond. Our results indicate that curdlan and PGA exist as a 1:2 molecular assembly, held together by hydrogen bonds between hydroxyls OH-2 and OH-4 of the polysaccharide units of curdlan and the carboxylic-/amide protons of PGA. This kind of molecular superstructure may be similar to the molecular system proposed recently for curdlan-βCD systems.²⁵

Figure 3.

MDM incubated with 150 ng mL⁻¹ curdlan nanoparticles from 0 to 48 hrs. Representative images of MDM uptake are shown at (a) 0 hr, (b) 2 hr, (c) 4 hr, (d) 8 hr, (e) 12 hr, (f) 24 hr, and (g) 48 hr. The red fluorescence arises from the TRITC labeled C-NPs, and the blue fluorescence arises from DAPI staining of nuclei. h) Quantification of intracellular uptake expressed as relative fluorescent units (RFU).

Figure 2.

¹H-¹H NOESY spectra (500 MHz, 25°C, mixing time: 100 ms) of a 1:2 mixture (wt%) of curdlan and PGA in DMSO-d₆.

In vitro effects of curdlan nanoparticles:

Data shown in Figure 3 are representative images of MDM post-incubation (2–48 hr) with C-NP, followed by quantification of intracellular uptake quantified by ImageJ. Data demonstrate a time-dependent increase in intracellular uptake of curdlan nanoparticles by MDM.

Toxicity of nanoassembly:

The cytotoxicity of C-NPs to MDM was assessed using an MTT assay (Figure 4). MDM incubated with 150 ng mL⁻¹ PGA, 150 ng mL⁻¹ Curdlan solution or 150 ng mL⁻¹ C-NPs for 24 hrs. No significant change in MDM viability was observed in the presence of PGA, Curdlan solution or C-NPs, relative to the untreated control.

Figure 4.

MDM incubated with 150 ng mL⁻¹ PGA, 150 ng mL⁻¹ Curdlan (Curd) solution or 150 ng mL⁻¹ C-NPs for 24 hrs. Viability was assayed using an MTT assay (n = 8 to 10, mean ± S.D).

Nanoassembley effects of cytokine gene expression in MDM:

A significant dose dependent increase in the IL-8 and TNF-α gene expression occurred at 75, 150, and 300 ng mL⁻¹ C-NPs at 24 hr (Figure. 5). IL-6 gene expression was significantly increased at 150 and 300 ng mL⁻¹ of C-NPs, while no effect was seen on IL-10 gene expression.

Figure 5.

Dose-response curve of C-NP on gene expression. MDM were incubated for 24 hr with 0 to 300 ng mL⁻¹ C-NP, RNA was isolated, and cytokine expression was measured using Q-PCR. Data are presented as the transcript accumulation index (TAI) = 2 ^−ΔΔC_T, n = 4, mean ± S.D). * p=0.05; ** p <0.001; **** p<0.0001.

Nanoassembley effects of cytokine release in MDM:

We next investigated the release of select pro-inflammatory cytokines and chemokines at 4 hr post-incubation with 150 ng mL⁻¹ of PGA solution, 150 ng mL⁻¹ curdlan solution or 150 ng mL⁻¹ of curdlan nanoparticles (Figure. 6) using BioLegend’s LEGENDplex™ bead-based immunoassays. A significant increase in the release of IL-lβ, MCP-1, TNF-α, IL-8, IL-12p70, IL-17, IL-18 and IL-23 occurred in the presence of 150 ng mL⁻¹ of curdlan solution compared to control which is expected as previously published ^{[16, 31–36]} Similarly, a significant increase in the release of IL-lβ, MCP-1, TNF-α, IL-8, IL-12p70, IL-17, IL-18 and IL-23 occurred in the presence of 150 ng mL⁻¹ of curdlan nanoparticles compared to control. No change in cytokine secretion occurred with PGA alone. When comparing the curdlan solution to the C-NP, there was significantly less cytokine production induced by the curdlan solution compared to the C-NP. These data suggest that curdlan in the C-NP is in the particulate form necessary to engage the dectin-1 receptor and activate macrophage ^[16].

Figure 6.

Effect of C-NPs on the release of cytokines and chemokines from MDM. MDM were incubated for 4 hr with 150 ng mL⁻¹ of PGA solution, 150 ng mL⁻¹ curdlan solution or 150 ng mL⁻¹ of curdlan nanoparticles The supernatant was collected and cytokines/chemokines were quantified using bead-based flow cytometry assay. Data are presented as pg m1⁻¹, n = 3, mean ± S.D. * significantly different compared to control; ** significantly different compared to C-NP. p=0.05

Discussion:.

A nanomedicine approach to immunotherapy has broad implications for therapeutic efforts against multiple diseases, including tuberculosis. Solutions of glucan incubated with M.tb infected macrophage, have been shown to significantly reduce the number of colony forming units (CFU) which is attributed to immune stimulation of the macrophage ^[34]. Based on these findings our laboratory as well as others have developed nanoparticles with glucan to significantly enhance the immune modulation of macrophage, with the ultimate goal of reducing intracellular M.tb. We have previously shown β-glucan as a ligand on PLGA-CS nanoparticles enhanced cytokine production from macrophage ^{[6, 11]} and reduced the number of CFU of Mycobacterium smegmatis, a mimetic of TB. While these nanoparticles are efficacious against M. smegmatis, we have found that synthesis of these nanoparticles is a complex, multi-step synthesis process that is not feasible for scale up processes and the synthesis process ultimately reduces the amount of drug that can be delivered. In the current study, we developed a simple curdlan (linear beta-1,3-glucan) based nanoparticle that is physically blended with PGA, thus bypassing the need for a PLGA-CS based nanoparticles ultimately reducing the number of steps in nanoparticle synthesis. Our C-NPs formed by blending of curdlan and PGA which eliminates the need for chemical modification or conjugation of curdlan that has been done previously ^{[5, 6, 10, 12, 13, 15, 37]} while maintaining the immunostimulatory capabilities of curdlan that is often lost with chemical modification with small molecules ^{[10, 15]}. Blending of curdlan and PGA has suitable processability and obtains the multicarboxylates originated from PGA. In addition to the expectation as an immune modulator originated from curdlan, C-NPs showed the swollen behavior in lower pH due to hydrogen bonding interference in lower pH, which suggests the possibility of C-NPs of targeting at a low pH and releasing bioactive therapeutics in the cellular compartment such as lysosome. The multicarboxylated C-NPs also can serve to form multiple-crosslinking domains with an organic crosslinker such as hydrogen peroxide activatable peroxalate or metal ions such as Ca²⁺, which would allow step-forward of C-NPs for better controlled drug release kinetics and activatable platform to other modality. Also, our NMR results showed that C-NPs is a nanoassembly mediated by polyvalent hydrogen bonding, whose structural integrity contributes to its stability in water (pH 7) for 9 days due to the robust hydrogen bonding. This further supports that curdlan and PGA form a stable complex which can be applicable to an in vivo model ^[19].

Curdlan binds Dectin-1 on macrophage to promote pro-inflammatory gene expression, intracellular ROS/RNS production and intracellular Ca²⁺ release ^{[32, 35, 36, 38–40]}. The proinflammatory cytokine TNF-α is important for M.tb eradication through induction of ROS/RNS as well as activation of phagocytes ^[41–43]. Glucans, including curdlan, are immune modulators of macrophage, enhancing the ability of macrophage to clear pathogens. We have demonstrated a role for C-NPs to stimulate the release of cytokines and modulate cytokine gene expression from MDM. It is likely that this enhanced production of TNF-α, IFN-γ, and IL-12p70 pro-inflammatory cytokines typical in activated M1 macrophage, will lead to an enhanced reduction in the intracellular pathogen burden. This study lays the foundation for a new treatment approach towards macrophage-based diseases, including TB, which may eliminate small molecule therapeutics that result in multi-drug resistant M.tb. Moreover, when combined with standard M.tb treatments, C-NPs will be an excellent adjuvant to orthogonally treat M.tb and reduce the course of treatment. Our future studies will investigate the ability of C-NPs to also delivery small molecules for TB therapeutics in anticipation of providing complete combination regimens for TB. Consequently, our immunotherapeutic approach, has the potential to harness the action of the innate immune system to eradicate M.tb.