Pluronic F127/lecithin PLGA nanoparticles as carriers of monocyte-targeted jakinibs: a potential therapeutic platform

Abstract

Aim: In this study, PLGA nanoparticles (PNPs) emulsified in Pluronic F127 (F127)/Lecithin (LEC) were designed to load Itacitinib (ITA), a selective JAK1 inhibitor, for targeting human monocytes.

Materials & methods: The physicochemical characteristics of empty and ITA-loaded F127/LEC PNPs were analyzed. The binding and internalization of NPs in leukocytes were evaluated. The effect of NPs on monocyte activation and JAK1 inhibition was assessed.

Results: F127/LEC PNPs were selectively bound and internalized by monocytes, sparing other leukocytes. ITA-F127/LEC PNPs significantly dampened monocyte activation. They also inhibited the monocyte's ability to promote T-cell proliferation and inhibited proinflammatory cytokine production.

Conclusion: ITA-loaded F127/LEC PNPs showed potential for monocyte-targeted therapy, offering new avenues for disease treatment.

Graphical Abstract

Plain language summary

Article highlights.

Nanoparticle characteristics

• The F127/LEC PNPs exhibited a uniform particle size distribution with an average size exceeding 150 nm.

• The encapsulation efficiency of Itacitinib (ITA) in F127/LEC PNPs was 50.59%.

• The nanoparticles maintained stability for 10 days at four Celsius degrees, with consistent size and ζ-potential.

Binding & internalization

• F127/LEC PNPs were selectively bound and were internalized by monocytes, sparing other leukocyte subpopulations.

• This selective uptake suggests the potential for targeted drug delivery to monocytes.

Inhibition of monocyte activation

• ITA-F127/LEC PNPs significantly reduced the release of proinflammatory cytokines (TNF-α, IL-12, IL-1β, and IL-10) in IFN-γ-stimulated monocytes.

• Both free and encapsulated ITA inhibited STAT1 phosphorylation in IFN-γ-stimulated monocytes, aligning with previous studies on JAK inhibitors.

Impact on T-cell proliferation

• ITA-F127/LEC PNPs inhibited the ability of monocytes to stimulate T-cell proliferation.

• This effect consistently reduced surface expression markers and proinflammatory cytokine production.

In vitro release studies

• PLGA nanoparticles displayed a biphasic release of ITA, with an initial burst release of 34% within 4 h, followed by a slower release over the next 10 h (∼40%).

• The Korsmeyer-Peppas model best described the release profile, indicating a Fickian diffusion pattern.

Conclusion

Therapeutic potential

• ITA-loaded F127/LEC PNPs showed promising therapeutic potential for targeting monocytes in diseases where they play a crucial role.

• The nanoparticles effectively suppressed monocyte activation and function in vitro, suggesting potential applications in various inflammatory diseases.

Targeted drug delivery

• Monocytes selectively bind and internalize F127/LEC PNPs, highlighting their potential as vehicles for targeted drug delivery.

• Encapsulation of ITA in these nanoparticles could increase local drug concentration while reducing systemic side effects.

Future research

• Further, in vivo research is essential to validate these findings and thoroughly assess the clinical applications of these nanoparticles.

• The project's next phase will focus on evaluating the biosafety, biodistribution, and in vivo therapeutic effectiveness of ITA-F127/LEC PNPs in murine models.

1. Introduction

Monocytes are bone marrow-derived cells that can differentiate into macrophages and dendritic cells. After receiving inflammatory signals, monocytes migrate to tissues to perform various functions, such as phagocytosis, antigen presentation and production of soluble mediators, including cytokines and chemokines. Monocytes are crucial in initiating and resolving inflammation and recruiting other immune system cells [1]. Monocytes have been implicated in the development and progression of various diseases, such as cancer [2,3], cardiovascular diseases [4] and autoimmune conditions [5,6], making them therapeutic targets of interest.

Some cytokines implicated in monocyte activation and function have been found to signal through the JAK/STAT pathways. These signaling pathways involve phosphorylation cascades for inducing the transcription of genes related to inflammatory response and monocyte survival, proliferation and differentiation. Various mutations and polymorphisms in the JAK and STAT genes have been described. JAK2 gain-of-function (GOF) mutations have been associated with myeloproliferative disorders [7] and JAK1 and JAK3 GOF mutations have been linked to hematologic malignancies including T-cell acute lymphoblastic leukemia and solid organ neoplasms such as breast cancer [8]. Additionally, STAT-3 GOF mutations have been found in patients with autoimmune lymphoproliferative disease [9]. STAT-4 polymorphisms confer an increased risk of developing rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) [10] and have been associated with severe SLE manifestations, such as renal disease [11]. Furthermore, the mRNA expression levels of the suppressor of cytokine signaling 1 (SOCS1), a negative regulator of JAK-STAT pathways, inversely correlate with disease activity in patients with SLE. These findings suggest that modulating the JAK/STAT signaling pathway could be valuable for treating different diseases.

Jak inhibitors (jakinibs) have been used to treat several types of cancer [12], as well as inflammatory [13,14] and autoimmune conditions [15,16]. However, despite their contribution to alleviating clinical manifestations, jakinibs can cause side effects, such as severe bacterial, fungal and viral infections, anemia, thrombocytopenia, neutropenia, hypercholesterolemia and thrombotic phenomena [17]. Encapsulation of jakinibs in nanoparticles (NPs) can increase their therapeutic effect [18,19] while reducing systemic side effects. Polymeric NPs stand out as excellent carriers of hydrophobic drugs, thus offering the possibility of improving their release rate [20,21]. These NPs are very versatile vesicles that can be assembled using various types of surfactants [22], including lecithin, a glycerophospholipid mixture composed mainly of phosphatidylcholine (PC), known for its amphipathic properties and low toxicity [23]. Hybrid NPs that combine lipids and biodegradable polymers leverage the individual components' physical, chemical and mechanical properties [24].

In a previous study, our group evaluated the physicochemical properties, stability and leucocyte uptake of different preparations of poly(lactic-co-glycolic acid) NPs (PNPs) assembled in the presence of different surfactants. PLGA is a highly biodegradable and biocompatible polymer composed of lactic and glycolic acid monomers. Although PLGA has intrinsic immunogenic properties [25], PLGA nanoparticles have been used in several clinical trials, serving as encapsulation tools for various drugs to treat cancer, diabetes and infections [26]. A combination of lecithin (LEC) and pluronic F127 (F127) as surfactants was used to promote the physicochemical stability of the nanoparticles and entrapped bioactive. PNPs assembled with F127 and LEC (F127/LEC PNPs) showed higher hardness and stability. Monocytes internalized them to a greater extent than other leukocyte subpopulations [27]. These results suggested that PNPs were a potential tool for encapsulating drugs that must specifically reach monocytes.

Since several pathologies, including autoimmune diseases such as RA, are characterized by altered monocyte phenotype and function and increased activation of the JAK-STAT pathway [28–30], this study proposed the design of F127/LEC-PLGA NPs to encapsulate Itacitinib (ITA), a Janus kinase (JAK)1-selective inhibitor, in order to specifically inhibit the JAK-STAT pathway in monocytes. This platform could represent a potential therapeutic tool for patients affected by RA and other autoimmune and inflammatory diseases.

2. Materials & methods

2.1. Reagents

RPMI1640 medium with Glutamax™ supplement (RPMI-1640), fetal bovine serum (FBS) and Dulbecco's phosphate-buffered saline (PBS) were purchased from Gibco^®-Life Technologies (Grand Island, NY, USA) and penicillin and streptomycin from Cambrex-BioWhittaker (Walkersville, MD, USA). Histopaque-1077, trypan blue and the polyacid polymer (D,L-lactic-co-glycolic acid) (PLGA) with a 50:50 ratio of lactic to glycolic acid and acid termination (PLGA-COOH) Resomer RG-503 (24,000–38,000 g/mol) were provided by Sigma Aldrich (St. Louis, MO, USA). Carboxyfluorescein succinimidyl ester (CFSE), 3,3′-dihexyloxycarbocyanine (DIOC6), propidium iodide (PI), Hoechst 33342 and LysoTracker™ Green DND-26 were purchased from Invitrogen (CA, USA). The LIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit, for excitation at 633 or 635 nm and L-glutamine were provided by Thermo Fisher Scientific (MA, USA). Vacutainer tubes with EDTA and BD Phosflow™ Perm Buffer IV were purchased from BD Biosciences (San Diego, CA, USA). FluorSave™ reagent was provided by Calbiochem (San Diego, CA, USA), Optylise C lysis buffer by Beckman Coulter (Brea, CA, USA) and Itacitinib by Cayman Chemical (Ann Arbor, MI, USA). Fluorochrome-conjugated monoclonal antibodies (mAbs) against CD45-PE-Cy7 (Clone HI30), p-STAT-1 (pY701)-Alexa Fluor^® 647 (Clone 4a) and CD86-FITC (Clone 2331) were purchased from BD Biosciences. Anti-CD69-PE-ECD (Clone TP1.55.3) mAb was provided by Beckman Coulter and anti-CD19-DR-APC-Cy7 (Clone SJ25C1) by Biolegend (San Diego, CA, USA). Lecithin (Research Products International Corp), Pluronic^® F127 (F127; HLB of 22), acetone and acetonitrile were HPLC-grade and purchased from Merck (Darmstadt, Germany).

2.2. Preparation of PNPs

PLGA PNPs were prepared via the nanoprecipitation method [31–33]. Initially, an organic phase was formed by dissolving 50 mg of PLGA and one wt% of Itacitinib in 2.5 ml of acetone. Subsequently, the organic phase was slowly added, at a rate of 0.1 ml/min, to a 15 ml aqueous solution containing one wt% surfactant (LEC, F127 and F127/LEC (30/70, w/w)), under constant agitation at 300 rpm. After the formation of the nanoparticle suspension, agitation was maintained for 48 h at room temperature to allow for the evaporation of the organic solvent. The resulting nanoparticle suspension underwent purification and concentration twice using a Digicen 21 R centrifuge (Orto-Alresa, Spain) at 10000 rpm or 9168 rotational centrifugal force (RCF) for 10 minutes at -4°C. Subsequently, the nanoparticles were resuspended in type 1 water with the assistance of a Branson SFX550, 550 W 20 kHz, 13 mm diameter ultrasound probe, applying 10% power for 10 seconds.

2.3. Characterization of nanoparticles

2.3.1. Nanoparticle size & ζ-potential

The hydrodynamic size (Z-average size) and ζ-potential of PNP suspensions were evaluated by dynamic light scattering (DLS) on a Zetasizer Pro instrument (Malvern Instruments Ltd, Malvern, UK). Nanoparticle suspensions were suitably diluted in deionized water and the Z-average size was determined by combining measurements at two single angles: 173° and 13°. For ζ-potential measuring, PNP suspensions were suitably diluted in deionized water (≈ 0.1%, m/v) and analyzed in a DTS 1070 Z-cell at 25°C. All measurements were done in triplicate for each sample.

2.3.2. Nanoparticle morphology

Nanoparticle suspensions were deposited on copper grids and stained with uranyl acetate to be analyzed by high-resolution transmission electron microscopy (HR-TEM) using a JEM-2100UHR electron microscope (JEOL Ltd. Tokyo, Japan) equipped with a 200-kV LaB6 filament. Nanoparticle analysis utilized SEM following the methodology outlined by Cruz et al. [27]. To conduct this analysis, we deposited nanoparticle suspensions on copper grids and stained them with uranyl acetate for the transmission mode of analysis; for the scanning mode, a drop of suspension was deposited on an aluminum foil without any staining procedure, using an Apreo 2 S LoVac field emission scanning electron microscope (Thermo Fischer Scientific) in STEM mode. The total number of counts reached 57,200 over an acquisition time of 60 seconds, with an average count rate of 1,063 cps at an acceleration voltage of 20 kV.

2.3.3. Encapsulation efficiency

The encapsulation efficiency of ITA in PNPs was determined and quantified by HPLC using an UltiMate 3000 HPLC/UHPLC chromatographic system (ThermoFisher Scientific, Karlsruhe, Germany), Hypersil Gold C18, 150 mm column, 25°C and Itacitinib (INCB039110) as reference standard. HPLC was done under the following conditions: injection volume of 10 μl, mobile phase (30 mM KH2PO4 in type I grade water: acetonitrile) in gradient runs, flow rate of 0.8 ml/min during 15 min (Supplementary Table S1) and detection wavelength of 310 nm (Diode Array Detector). A calibration curve for ITA quantification was prepared with 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0 and 100 ppm calibration standards that were filtered through PVDF membranes with a 0.22 μm pore size and packaged in chromatographic vials (Supplementary Figure S1). Nanoparticle samples were prepared using 300 μl of PNP-derived supernatant, obtained during PNP washing and 1200 μl of acetonitrile. These sample suspensions were filtered through PVDF membranes with a 0.22 μm pore size and packaged in chromatographic vials. The encapsulation efficiency (EE) of ITA was calculated as follows (Equation 1):

$$\begin{array}{c}\text{EE}(\%)=\\ \frac{\text{Itacitinib added}\left(\text{mg}\right)-\text{Itacitinib in the supernatant}\left(\text{mg}\right)}{\text{Itacitinib added}\left(\text{mg}\right)}\times 100\end{array}$$ (1)

2.4. Stability of the nanoparticles

PNP suspensions were kept at 4°C. Their physicochemical features (Z-average size, PDI and ζ-potential) were evaluated after 1, 2, 4, 6, 8 and 10 days of storage as described in section 2.3.1.

2.5. In vitro release study

The Itacitinib released from the NPs of PLGA was evaluated by simulating physiological conditions. The methodology described by Pachiyappan et al. was used with minor modifications [34]. Briefly, 15 mg of NPs resulted in 1.5 ml of phosphate buffer (DPBS1X; pH 7,4) solution and 14 KDa dialysis bags, Pd 2,3 nm, were loaded. Once the bag was sealed, it was introduced at 30 ml of 1× DPBs solution (2% Tween 80), 37°C and 300 rpm. According to the analysis time, 0.5 ml of solution was removed and compensated with 0.5 ml of DPBS1X solution (2%, Tween 80). The released Itacitinib was analyzed using HPLC and using the method described previously (Hypersil Gold C18, 150 mm column. Gradient. ACN – KH2PO4 50 mm in H2O. 15 min and 25°C. λ = 310 nm). Different mathematical models were used to calculate the release data, as described below:

The Korsmeyer-Peppas model explains the release of drugs from a polymeric system [35,36]. The Kors-Peppas Equation 2 is:

$$Q_t=K_{\mathit{KP}}{t}^n$$ (2)

Where Qt: Drug fraction released in time, KKP: Korsmeyer-Peppas Kinetic Constant, n: exponent of diffusion or release, t: time in h.

2.6. Fourier transform infrared spectroscopy of PLGA-LEC/F127 NPs loaded with Itacitinib

NPs were analyzed using a PerkinElmer (Waltham, Massachusetts, USA) Spectrum two Fourier transform infrared spectrophotometer with attenuated total reflectance (UATR) modulus between 550 and 4000 cm^-1 at a resolution of 4 cm^-1.

2.7. Nanoparticle binding to leukocytes

Peripheral blood samples from healthy individuals were incubated with F127/LEC PNPs loaded with the fluorescent tracer curcumin for one hour at 37°C. Afterward, cells were labeled with anti-CD45-PE-Cy7 mAb and stained with LysoTracker at 37°C for 30 min. Erythrocytes were then lysed by adding 250 μl of Optilyse C lysis buffer and 250 μl of distilled water. Finally, cells were acquired on a LSR Fortessa flow cytometer and FACS DIVA software flow cytometer to analyze the binding of PNPs to different leukocyte subpopulations.

2.8. Isolation of peripheral blood mononuclear cells (PBMCs)

PBMCs were isolated from EDTA-anticoagulated peripheral blood from healthy individuals by Histopaque gradient (1.077 g/ml) centrifugation at 900 × g for 30 min at 21°C. PBMCs were washed twice with PBS at 300 × g for 10 min and resuspended in RPMI1640 supplemented with penicillin (100 U/ml)/streptomycin (100 μg/m) and 10% iFBS. Cell count and viability were determined by trypan blue dye exclusion using a Neubauer chamber.

2.9. Nanoparticle uptake by monocytes

A PBMC suspension (containing 5 × 10⁵ CD14⁺ cells in 500 μl of RPMI1640 supplemented with 0.5% iFBS) was seeded onto 12 mm glass coverslips (Marienfeld, Lauda-Königshofen, Germany), previously treated with poly-L-lysine, and incubated for 2 h at 37°C and 5% CO2. The coverslips were washed with PBS containing 0.5% iFBS to remove non-adherent cells. Next, adherent cells were incubated with 500 μl of RPMI1640 supplemented with 10% iFBS at 37°C and 5% CO2. After 24-h incubation, Nile Red-loaded F127/LEC PNPs and 2 μM LysoTracker were added, and cells were incubated for another hour. Subsequently, 400 μl of culture medium was carefully removed, and cells were washed with 400 μl of PBS and stained with Hoechst 33342 (1:5000) for 30 min at RT. Excess Hoechst 33342 was removed, and cells on coverslips were preserved with FluorSave™ solution (Calbiochem, San Diego, CA). Finally, coverslips were observed under an inverted fluorescence microscope Axio Vert.A1 (Zeiss, Oberkochen, Germany) using the Zen image analysis software (Zeiss, Oberkochen, Germany). Images were analyzed with the ImageJ software (Rasband WS, National Institute of Health, Bethesda, MD, USA) to assess the colocalization of LysoTracker and Nile Red signals, using the Pearson's correlation coefficient, in at least five randomly selected cells in each image.

2.10. Isolation of monocytes & T-cells

Monocytes and T-cells were enriched from PBMC suspensions by negative selection using a BD FACSAria™ III Cell Sorter (BD Biosciences). Briefly, 10 × 10⁶ PBMCs were resuspended in 1 ml of RPMI with 2% EDTA and labeled with anti-CD19-APC-Cy7 mAb. T-cells were selected as CD19^neg cells and monocytes according to size (FSC-A) and granularity (SSC-A) parameters. Monocytes and T-cells were sorted and collected on RPMI 1640 in plastic tubes. Cell purity (≥90%) was assessed by flow cytometry.

2.11. Monocyte activation

Monocytes (2 × 10⁵ cells) were cultured in 96-well U-bottom plates at 37°C and 5% CO2 in 200 μl of RPMI 1640 supplemented with penicillin (100 U/ml)/streptomycin (100 μg/ml) and 10% iFBS, and stimulated with 2 ng/ml IFN-γ for 24 h in the presence of free ITA, empty FL127/LEC PNPs, or ITA-loaded FL127/LEC PNPs (ITA-F127/LEC PNPs) at a concentration equivalent to 1200 ng/ml of ITA. Subsequently, the cells were washed with PBS and stained with anti-CD69-PE and anti-CD86-FITC mAbs for 30 min at 37°C, washed, and acquired on a LSR Fortessa flow cytometer, and the data were analyzed with FlowJo vX software (BD Life Science, Ashland, OR, USA. Changes in CD69 MIF and CD86 MIF were evaluated.

After culture, supernatants were collected and stored at -20°C until cytokine quantification. The supernatants were then thawed to measure TNF-α, IL-12, IL-1β, and IL-10 concentrations using xMAP microspheres, the Luminex^® 200™ System, and xPONENT^® software, following the manufacturer's instructions.

2.12. STAT1 phosphorylation

Monocytes (2 × 10⁵ cells) were cultured in 96-well U-bottom plates with 200 μl of RPMI supplemented with penicillin (100 U/ml)/streptomycin (100 μg/ml) and 10% iFBS at 37°C and 5% CO2. Cells were then exposed for one hour to ITA, empty F127/LEC PNPs, or ITA-F127/LEC PNPs at a concentration equivalent to 1200 ng/ml ITA. Next, 2 ng/ml IFN-γ was added, and cells were incubated for 15 min. Afterward, cells were fixed with BD Phosflow™ Lyse/Fix Buffer (BD Biosciences) and permeabilized with BD Phosflow™ Perm Buffer IV (BD Biosciences), following the manufacturer's instructions, stained with an anti-STAT1(pY701)-Alexa Fluor^® 647 and acquired on a LSR Fortessa flow cytometer to evaluate the STAT1(pY701) MFI.

2.13. T-cell proliferation

T cells were stained with one microliter of CFSE (5 μM in PBS with 0.1% bovine serum albumin [BSA]) for 15 min at 37°C in the dark. Cells were then centrifuged at 600 × g for 10 min, the supernatant was removed, RPMI supplemented with 10% iFBS was added, and cells were incubated another 40 min at 37°C and 5% CO2 to remove excess CFSE dye. Likewise, monocytes (1 × 10⁵ cells) were cultured in U-bottom 96-well plates for 4 h in RPMI supplemented with penicillin (100 U/ml)/streptomycin (100 μg/ml) and 0.5% iFBS to promote cell adhesion. Subsequently, adherent monocytes were incubated with ITA, empty FL127/LEC PNPs, or ITA-F127/LEC PNPs at a concentration equivalent to 1200 ng/ml of ITA. After 1 h, PNPs were removed by washing with PBS, and monocytes were co-cultured for 72 h with CFSE-labeled autologous T cells stimulated with 2 μg/ml phytohemagglutinin (PHA) in RPMI supplemented with penicillin (100 U/ml) / streptomycin (100 μg/ml) and 5% iFBS. The percentage of proliferating cells and staining index were assessed by flow cytometry according to the CFSE MFI reduction using the FlowJo VX software.

After culturing, supernatants were collected and stored as indicated to measure IL-2, IFN-γ, and IL-17 concentrations using xMAP microspheres, the Luminex^® 200™ System, and xPONENT^® software, following the manufacturer's instructions.

2.14. Statistical analysis

Comparisons between cells exposed or not to ITA-loaded F127/LEC PNPs were analyzed with the one-tailed Wilcoxon test. The Pearson correlation coefficient evaluated the colocalization of fluorescence signals on confocal fluorescence microscopy. All statistical analyses used GraphPad Prism version 9, GraphPad Software (MA, USA, www.graphpad.com). The level of significance was defined as *p < 0.05.

3. Results

3.1. Characterization of nanoparticles

Dynamic light scattering analyses showed that empty and ITA-loaded F127/LEC PNPs exhibited an average size below 200 nm and ζ-potential values of -45.3 ± 1.9 mV and -30.96 ± 1.6 mV, respectively (Table 1).

Table 1.

Physicochemical properties of F127/LEC PNPs.

Nanoparticle	Load	Size (nm) Meana	ζ-Potential (mV)	PDI
F127/LEC PNPs	Empty	181 ± 5a	-45.3 ± 1.9a	0.11 ± 0.03a
	Itacitinib	193 ± 2b	-30.96 ± 1.6b	0.19 ± 0.01b

^aParticle size (z average).

Different letters indicate a statistically significant difference (p < 0.05).

PDI: Polydispersity index.

Under electron microscopy, ITA-F127/LEC PNPs were observed as spherical and smooth-surfaced particles with an average size below 200 nm (Supplementary Figure S2). Figure 1 shows a high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image of empty and Itacitinib-loaded PLGA-LEC/F127 nanoparticles. The image reveals a uniform particle size distribution, with an average particle size exceeding 150 nm, consistent with dynamic light scattering (DLS) measurements. The homogeneous contrast observed within the nanoparticles confirms their structural integrity, and the variation in contrast within individual particles indicates the presence of a core-shell structure, particularly in the empty nanoparticles.

Figure 1.

SEM of ITA-F127/LEC PNPs. (A) Low magnification high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image of PLGA-LEC-F127 NPs. (B) Electronic scanning micrography of PLGA-LEC/F127 NPs loaded with Itacitinib.

Supplementary Table S2 provides the atomic composition data from energy-dispersive x-ray spectroscopy (EDS) of Itacitinib-loaded nanoparticles. Fluorine (F) within the nanoparticles suggests successfully incorporating the Itacitinib drug, as indicated by the F signal corresponding to the Itacitinib molecule.

The encapsulation efficiency of ITA in F127/LEC PNPs was 50.59%.

$$\begin{array}{c}\mathit{Itacitinib }\left(\mathit{mg}\right){}_{\mathit{supernatant}}=\\ \frac{(1.5\mathit{ }\text{ml}\times 138.353\mathit{ }\text{ppm})}{0.3\mathit{ }\text{ml}}\times 0.012\mathit{ }\text{L}=0.830118\mathit{ }\text{mg}\end{array}$$

$$\mathit{EE}(\%)=\frac{\left(1.68-0.830118\right)\text{mg}}{1.68\text{ mg}}\times 100=50.59\%$$

Moreover, PNP size and ζ-potential remained constant for 10 days at 4°C, highlighting their stability under the storage conditions used here (Supplementary Figure S3).

3.2. In Vitro Release Studies of Itacitinib Loaded in PLGA-LEC/F127 NPs

PLGA nanoparticles displayed a biphasic release of Itacitinib, with an initial burst releasing 34% of the drug within 4 h, followed by a slower release over the subsequent 10 h (∼40%). This rapid initial release likely results from the drug adhering to the polymer surface. In contrast, the sustained release phase is attributed to the gradual degradation of PLGA in the dissolution medium. Various kinetic models were applied to the in vitro release data to elucidate the release mechanism of Itacitinib from PLGA nanoparticles. The Korsmeyer-Peppas model best described the release profile over 8 h, indicating a Fickian diffusion pattern with an n value of less than 0.45 (Supplementary Table S3). The size and morphology of the PLGA-LEC/F127 nanoparticles proved effective for the controlled release of Itacitinib, showing an initial rapid release phase lasting 5 h, followed by a sustained release phase. This biphasic release model ensures the prolonged delivery of the drug. The kinetic parameters were determined using a linear regression model, with results presented graphically in Supplementary Figure S4 and summarized in Supplementary Table S3.

3.3. FTIR analysis of PLGA-LEC/F127 NPs loaded with Itacitinib

Supplementary Figure S5 presents the FTIR spectra of both empty and Itacitinib-loaded PLGA-LEC/F127 nanoparticles. The spectrum of empty NPs shows a strong peak at 1750 cm^-1, which is attributed to carbonyl stretching (C=O), alongside peaks at 1087 cm^-1 (C-O-C stretching), 1057 cm^-1 (C-CH3 stretching), and 1454 cm^-1 (C-H stretching of methyl groups). Bands at 1385 and 877 cm^-1 correspond to in-plane and out-of-plane O-H bending vibrations, respectively.

The 757 cm^-1 band is attributed to out-of-plane C-H bending, while the 1184 cm^-1 band corresponds to terminal hydroxyl groups of PLGA chains, and the 1268 cm^-1 peak is associated with ester groups of the polymer. The spectrum's similarity to characteristic peaks reported for PLGA powder by Akl et al. [37] confirms the integrity of the polymer during nanoparticle processing.

In contrast, the spectrum of Itacitinib-loaded nanoparticles did not exhibit the characteristic absorption bands of Itacitinib. due to its low concentration compared with the polymer. However, notable amide (N-H) stretches were observed at 3400–3200 cm^-1 and amide II (N-H) bands at 1580–530 cm^-1 as well as a slight signal at 1637 cm^-1 related to symmetric stretching of the C=C bond from Itacitinib.

3.4. Monocytes preferentially took up F127/LEC PNPs without affecting their viability

The binding and internalization of curcumin-loaded F127/LEC PNPs by human circulating leukocytes were assessed by flow cytometry. The fluorescent curcumin signal increased predominantly in monocytes compared with neutrophils and lymphocytes (Figure 2A & B). Additionally, internalization of F127/LEC PNPs by leukocyte subpopulations was assessed as acidification of intracellular compartments (LysoTracker MIF). LysoTracker MIF increased significantly in monocytes cultured with F127/LEC PNPs (Figure 2C). Furthermore, exposure to F127/LEC PNPs did not alter cell membrane integrity or mitochondrial membrane potential (Supplementary Figure S6). These results evidenced that F127/LEC PNPs were mainly localized around and internalized by monocytes. Therefore, F127/LEC PNPs could be a valuable platform for encapsulating monocyte-targeted compounds.

Figure 2.

Binding and internalization of curcumin-loaded F127/LEC PNPs in human circulating leukocytes. Peripheral blood samples from healthy individuals were incubated with curcumin-loaded F127/LEC PNPs for 1 h at 37°C and labeled with anti-CD45-PE-Cy7 mAb and LysoTracker. After erythrocyte lysing, cells were analyzed by flow cytometry. (A) Representative dot plots showing curcumin fluorescent signal in neutrophils, monocytes, and lymphocytes discriminated by SSC-A. (B & C) Curcumin MFI and Lysotracker MFI of monocytes, neutrophils, and lymphocytes in cell suspensions incubated with and without (wo) curcumin-loaded F127/LEC PNPs. Wilcoxon test, *p < 0.05. (n = 4 independent experiments).

PNP: Poly(lactic-co-glycolic acid) nanoparticle.

Confocal fluorescence microscopy confirmed the internalization of F127/LEC PNPs by monocytes. Indeed, colocalization of Nile Red and Lyso-Tracker signals (Pearson correlation coefficient = 0.83) was observed in monocytes incubated for 1 h with Nile Red-loaded PNPs and stained with LysoTracker (Figure 3).

Figure 3.

Binding and internalization of F127/LEC PNPs in human circulating monocytes. Monocytes adhered to coverslips were cultured with RPMI1640 supplemented with iFBS at 37°C for 24 h before adding Nile Red-loaded F127/LEC PNPs and 2 μM LysoTracker. After PBS washing, cells were stained with Hoechst 33342 preserved with FluorSave™ solution and observed under an inverted fluorescence microscope Axio Vert.A1 using a Zen image analysis software. Images were evaluated with the ImageJ software. (A) Confocal fluorescence microscopy images of Hoechst 33342-stained nuclei (blue), Nile Red-loaded NPs (red), Lyso-Tracker-labeled acidic compartments (green), and merged signals (yellow). Inset: correspond to the amplification of merge signals demarcated in white.

3.5. ITA-loaded F127/LEC PNPs reduce CD69 & CD86 expression levels on monocytes & inhibit the production of proinflammatory cytokines

The ability of ITA-loaded F127/LEC PNPs to modulate monocyte activation was determined in monocyte cultures stimulated with 2 ng/ml of IFN-γ in the presence of ITA, empty PNPs, and ITA-F127/LEC PNPs for 24 h. CD69 and CD86 expression was significantly (p < 0.05) increased in IFN-γ-stimulated monocytes compared with unstimulated cells. Furthermore, in contrast to empty NPs, ITA and ITA-loaded PNPs significantly (p < 0.05) decreased the stimulatory effect of IFN-γ on monocytes, thus resulting in inhibition percentages of approximately 25% and 10% in the MFI of membrane CD86 and CD69, respectively (Figure 4).

Figure 4.

Effect of ITA-F127/LEC PNPs on monocyte activation. Monocytes, cultured in 96-well U-bottom plates with RPMI supplemented with penicillin/streptomycin and iFBS at 37°C, were stimulated with 2 ng/ml IFN-γ for 24 h in the presence of ITA, empty FL127/LEC PNPs, or ITA-F127/LEC PNPs at a concentration equivalent to 1200 ng/ml of ITA. Cells were then stained with anti-CD69-PE-ECD and anti-CD86-FITC mAbs, washed, and analyzed by flow cytometry. (A) CD69 MFI and (B) CD86 MFI of IFN-γ-stimulated monocytes were compared with the respective MFI of IFN-γ-stimulated monocytes previously exposed to different treatments.

*p < 0.05; one-tailed Wilcoxon test. n = 5 independent experiments.

ITA: Itacitinib; PNP: Poly(lactic-co-glycolic acid) nanoparticle.

TNF-α, IL-12, IL-1β, and IL-10 concentrations increased in monocytes stimulated with IFN-γ compared with unstimulated cells. ITA-F127/LEC PNPs and free ITA reduced the release of these cytokines by these cells (Supplementary Figure S7).

3.6. ITA & ITA-F127/LEC PNPs inhibited STAT1 phosphorylation in human monocytes

As expected, STAT1(pY701) levels significantly increased (p < 0.05) in monocyte-enriched cultures stimulated with IFN-γ for 15 min. However, STAT1(pY701) levels significantly decreased (p < 0.05) in IFN-γ-stimulated monocytes previously exposed for one hour to ITA or ITA-F127/LEC PNPs reaching inhibition percentages of 84% and 72%, respectively (Figure 5).

Figure 5.

Effect of ITA-F127/LEC PNPs on STAT1 phosphorylation of monocytes. Monocytes, cultured in 96-well U-bottom plates with RPMI supplemented with penicillin/streptomycin and iFBS at 37°C, were treated with ITA, empty F127/LEC PNPs, or ITA-loaded-F127/LEC PNPs for one hour before stimulation with IFN-γ for 15 min. Cells were then fixed with BD Phosflow™ Lyse/Fix Buffer, permeabilized with BD Phosflow™ Perm Buffer IV, stained with an anti-STAT1(pY701)-Alexa Fluor^® 647, and analyzed by flow cytometry. (A) Representative histograms showing the STAT1(pY701) MFI changes under different treatments. (B) STAT1(pY701) MFI of IFN-γ-stimulated monocytes was compared with the STAT1(pY701) MFI of IFN-γ-stimulated monocytes previously exposed to different treatments.

*p < 0.05; one-tailed Wilcoxon test. n = 5 independent experiments.

ITA: Itacitinib; PNP: Poly (lactic-co-glycolic acid) nanoparticle.

3.7. ITA-F127/LEC PNPs negatively modulate the monocyte's ability to promote T-cell proliferation & inhibit proinflammatory cytokine production

Monocytes pretreated for 1 h with ITA, F127/LEC PNPs, and ITA-F127/LEC PNPs were co-cultured with CFSE-labeled autologous T cells in the presence of PHA for 72 h. Flow cytometry analyses showed that the percentage of proliferating T cells decreased significantly (p < 0.05) in co-cultures previously exposed to free or encapsulated ITA (Figure 6).

Figure 6.

ITA-F127/LEC PNPs affect the monocyte's ability to promote T-cell proliferation. Monocytes, cultured in 96-well U-bottom plates, were treated for 1 h with 1200 ng/ml ITA, empty FL127/LEC PNPs, or ITA-F127/LEC PNPs at a concentration equivalent to 1200 ng/ml of ITA. After PBS washing for removing NPs, pretreated monocytes were co-cultured with CFSE-labeled autologous T cells and 2 μg/ml PHA in RPMI supplemented with penicillin/streptomycin and 5% iFBS at 37°C for 72 h. (A) Representative histograms showing the percentage of proliferating T cells under different treatments. (B) The percentage of proliferating T cells in PHA-stimulated co-cultures was compared with that of proliferating T cells under different treatments.

*p < 0.05; one-tailed Wilcoxon test. n = 5 independent experiments.

ITA: Itacitinib; PHA: Phytohemagglutinin; PNP: Poly (lactic-co-glycolic acid) nanoparticle.

IL-2, IFN-γ, and IL-17 concentration significantly increased in monocytes co-cultured with autologous T lymphocytes stimulated with PHA. The previous treatment of monocytes with ITA-F127/LEC PNPs and free ITA significantly reduced the release of these cytokines (Figure 7).

Figure 7.

Free and encapsulated Itacitinib modulates cytokines production. Monocytes from healthy controls incubated for one-hour empty F127/LEC PNPs, free Itacitinib, or encapsulated Itacitinib NPs (concentration equivalent to 400 ng/ml ITA) and co-cultured with autologous T lymphocytes stimulated or not with PHA. After 72 h, culture supernatants were collected to measure levels of (A) IL-2, (B) IFN-γ and (C) IL-17 by a Luminex bead-based multiplex assay. Three independent experiments, Wilcoxon (one-tailed) *p < 0.05.

4. Discussion

Monocytes and macrophages play a crucial role in the immune system, initiating, developing, and resolving various inflammatory disorders. Given their immunological relevance, these cells have become a target of interest for diagnosing and treating various diseases. Among potential immunomodulators, jakinibs constitute a promising group of drugs. However, the cell selectivity of these inhibitors and the need to reduce the adverse effects related to their use are key aspects to be considered. In the present study, we proposed to encapsulate jakinibs in NPs, specifically targeting monocytes to avoid compromising the function of other immune system cells. To this end, F127/LEC PNPs were designed to encapsulate the JAK1 inhibitor, ITA, as a platform capable of specifically interacting with monocytes. The effects of free and NP-encapsulated ITA on monocytes were compared. The results suggested that ITA-F127/LEC PNPs could be a valuable monocyte-targeted therapeutic platform, as they effectively inhibited monocyte activation and their ability to promote T-cell proliferation in vitro. Although free ITA and the encapsulated one have similar inhibitory effects, the encapsulated ITA-F127/LEC PNPs might provide the specificity not observed with free ITA, avoiding possible side effects. However, in vivo, studies are essential to confirm these in vitro findings.

The physicochemical characteristics of nanoparticles determine their function and interaction with different cell types [20]. Polymeric formulations, widely used in NP engineering, favor controlled release of the encapsulated compound, as degradation of the polymer can be tightly controlled [1]. PLGA is a polymer with a backbone composed of lactic and glycolic acid monomers. These monomers can be quickly metabolized through the Krebs cycle, so they are considered highly biodegradable and biocompatible. Indeed, the FDA and the EMA have approved their use in various formulations for clinical purposes [38]. One of the most widely used strategies to improve the properties of PNPs is the use of different synthetic surfactants such as polyvinyl alcohol (PVA), polysorbates such as Tween 80, and some poloxamer such as Pluronic^® F127 during their production. Although this last surfactant is the most widely used due to its low toxicity, it still shows undesirable effects in animal models, such as hypertriglyceridemia and hypercholesterolemia [39]. Due to those side effects, using emulsifying agents that are more compatible and safer for living organisms-preferably of natural origin- as lecithin is necessary. It contains 70% phosphatidylcholine (PC), 7%–11% phosphatidylethanolamine (PE), as well as some fatty acids such as linoleic, palmitic, and oleic acids [40]. Here, PNPs were produced in the presence of the surfactants F127 and lecithin, resulting in F17/LEC PNPs with an average size of <200 nm, potential ζ of -30.96 ± 1.6 mV, and stable under storage conditions at four Celsius degrees (Supplementary Figure S3). Furthermore, these PNPs were shown not to affect cell membrane integrity or mitochondrial membrane potential (Supplementary Figure S6), confirming their non-cytotoxic nature and usefulness for encapsulating and delivering drugs in humans.

Lipid-based NPs are frequently used formulations to target compounds to monocytes [1]. F127/LEC PNPs were preferentially bound and internalized in monocytes compared with neutrophils and lymphocytes. Previous studies demonstrated that macrophages efficiently uptake PC-coated liposome-like NPs through the scavenger receptor CD36, a receptor involved in the uptake of oxidized low-density lipoproteins [41,42]. Because PC is the major component of lecithin, CD36 could mediate the uptake of F127/LEC PNPs by monocytes.

Free and encapsulated ITA reduced the expression of activation markers CD86 and CD69 on IFN-γ-stimulated monocytes and decreased monocytes' ability to support autologous T-cell proliferation. In contrast, empty F127/LEC PNPs did not exert this effect, indicating that the empty PNPs themselves did not interfere with monocyte activation, but rather, the encapsulated ITA did. These results align with a previous study on tofacitinib, a JAK3 inhibitor that decreased CD80 and CD86 expression on LPS-treated monocyte-derived dendritic cells and their ability to stimulate T-cells [43].

Free and encapsulated ITA inhibited proinflammatory cytokine production (TNF-α, IL-12, IL-1β, and IL-10) by monocytes treated with IFN-γ. A previous study demonstrated that two different JAK inhibitors, AG490 (a specific inhibitor of JAK2) and ruxolitinib (an inhibitor of JAK1 and JAK2), attenuated cytokine production by monocyte-derived-macrophages (MDMs) stimulated by cigarette smoke extract (CSE) alone or in combination with lipopolysaccharide (LPS) [44]. Similarly, Barone et al. demonstrated that ruxolitinib inhibited the secretion of pro/anti-inflammatory cytokines by monocytes from myelofibrosis patients treated with LPS in vitro [45]. These findings underscore the potential use of Jakinibs to modulate proinflammatory cytokine production.

Furthermore, we observed a significant increase in IL-2, IFN-γ, and IL-17 concentration in monocytes co-cultured with autologous T lymphocytes stimulated with PHA. Neustock et al. demonstrated that Mono Mac 6 (a human monocytic cell line) and human monocytes produce cytokines in response to PHA treatment [46]. Another study showed that treating PBMCs with PHA increased STAT and p-STAT levels, suggesting that PHA could activate the JAK/STAT pathway [47]. Consistent with these results, we found that pretreatment of monocytes with ITA-F127/LEC PNPs and free ITA significantly reduced the release of these cytokines.

Free and encapsulated ITA also inhibited STAT1 phosphorylation in IFN-γ-stimulated monocytes. Similarly, Tucci et al. showed a significant reduction of STAT1 phosphorylation in IFN-α-stimulated monocytes from patients with RA given Baricitinib (a JAK1/JAK2 inhibitor) for 4–12 weeks compared with monocytes from untreated patients [48].

Finally, it is essential to note that F127/LEC PNPs were ingested mainly by monocytes compared with other circulating leukocyte subpopulations, so they could be used as vehicles to deliver drugs to monocytes. Indeed, ITA encapsulated in F127/LEC NPs inhibited monocyte activation and function in vitro, thus suggesting that these NPs could have therapeutic applications in several diseases in which monocytes play a central role. Moreover, the internalization of NPs by monocytes could favor tissue accumulation of particles because, after NP internalization, monocytes can respond to chemokines released in the foci of inflammation and infection [49]. However, the results observed in vitro required the support of in vivo tests. Intravenous administration of encapsulated ITA to animal models is expected to protect the drug from interaction with plasma proteins such as complement factors and immunoglobulins and from enzymatic degradation. In addition, encapsulation helps ITA to be targeted to monocytes, thus increasing its local concentration while reducing side effects attributed to high systemic concentrations of the drug or its interactions with other immune system cells or tissues. In the next phase of this project, our primary objective is to evaluate the biosafety, biodistribution comprehensively, and in vivo therapeutic effectiveness of ITA-F127/LEC PNPs in murine models. Building upon previous research demonstrating the biocompatibility and promising biomedical potential of these NPs, we maintain an optimistic outlook.

5. Conclusion

Our study revealed that F127/LEC PNPs exhibit specific binding and internalization by monocytes. Efficient encapsulation of the JAK1 inhibitor, ITA, was achieved in these nanoparticles. ITA-F127/LEC PNPs effectively suppressed monocyte activation, as evidenced by reduced surface expression markers and proinflammatory cytokine production, as well as their ability to stimulate T-cell proliferation in vitro. These results indicate the promising therapeutic potential of these nanoparticles for targeting monocytes in diseases where they play a crucial role. However, further in vivo research is essential to validate these findings and thoroughly assess the clinical applications of these nanoparticles.

Funding Statement

The work was funded by MINCIENCIAS through the Contingent Recovery Financing Contract No. 925 of 2019, signed on 19 December 2019, for the project: “Polymeric nanoparticles conjugated with mannose and lectins for the encapsulation of selective inhibitors of Janus kinases directed towards monocytes for the alternative treatment of auto immunities”. Code 111584467267, and the University of the Amazonia by agreement letter 060620. K Álvarez was a recipient of a doctoral scholarship from Universidad de Antioquia “Fondo becas doctoral UdeA”.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17435889.2024.2415877

Author contributions

LF Giraldo, VH Orozco, G Vásquez and M Rojas-López were involved in planning and supervised the work, and L Álvarez and JT Cruz manufactured the samples and characterized them, processed the experimental data, performed the analysis, drafted the manuscript and designed the figures. All authors discussed the results and commented on the manuscript.

Financial disclosure

The work was funded by MINCIENCIAS through the Contingent Recovery Financing Contract No. 925 of 2019, signed on 19 December 2019, for the project: “Polymeric nanoparticles conjugated with mannose and lectins for the encapsulation of selective inhibitors of Janus kinases directed towards monocytes for the alternative treatment of auto immunities”. Code 111584467267, and the University of the Amazonia by agreement letter 060620. K Álvarez was a recipient of a doctoral scholarship from Universidad de Antioquia “Fondo becas doctoral UdeA”. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

This study, titled “Nanopartículas Poliméricas conjugadas con manosa y lectinas para la encapsulación de inhibidores selectivos de las Janus kinasas dirigidas hacia monocitos para el tratamiento alternativo de autoinmunidades”, was conducted in accordance with the highest ethical standards. The research protocol was reviewed and approved by the Ethics Committee of the Instituto de Investigaciones Médicas under Act No. 009, with approval granted on 9 May 2019.

This study adhered strictly to these ethical principles, ensuring the protection and well-being of all participants. No amendments to the original protocol were made during the study. All participants were volunteers who signed the informed consent form.