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. 2024 Jan 17;7(8):4856–4866. doi: 10.1021/acsabm.3c00985

Uptake of Cyclodextrin Nanoparticles by Macrophages is Dependent on Particle Size and Receptor-Mediated Interactions

Shreya S Soni †, Kenneth M Kim †,‡, Biplab Sarkar †, Christopher B Rodell †,*
PMCID: PMC11252246  NIHMSID: NIHMS1966284  PMID: 38231485

Abstract

graphic file with name mt3c00985_0007.jpg

Physiochemical properties of nanoparticles, such as their size and chemical composition, dictate their interaction with professional phagocytes of the innate immune system. Macrophages, in particular, are key regulators of the immune microenvironment that heavily influence particle biodistribution as a result of their uptake. This attribute enables macrophage-targeted delivery, including for phenotypic modulation. Saccharide-based materials, including polyglucose polymers and nanoparticles, are efficient vehicles for macrophage-targeted delivery. Here, we investigate the influence of particle size on cyclodextrin nanoparticle (CDNP) uptake by macrophages and further examine the receptor-mediated interactions that drive macrophage-targeted delivery. We designed and synthesized CDNPs ranging in size from 25 nm to >100 nm in diameter. Increasing particle size was correlated with greater uptake by macrophages in vitro. Both scavenger receptor A1 and mannose receptor were critical mediators of macrophage-targeted delivery, inhibition of which reduced the extent of uptake. Finally, we investigated the cellular bioavailability of drug-loaded CDNPs using a model anti-inflammatory drug, celastrol, which demonstrated that drug bioactivity is improved by CDNP loading relative to free drug alone. This study thus elucidates the interactions between the polyglucose nanoparticles and macrophages, thereby facilitating their application in macrophage-targeted drug delivery that has applications in the context of tissue injury and repair.

Keywords: Nanomedicine, nanoparticle, macrophage, cyclodextrin, receptor-mediated uptake

1. Introduction

Macrophages not only support the homeostatic equilibrium in various tissues1 but also orchestrate the immune sequelae that underlies disease and tissue healing. Macrophages in the setting of disease do not necessarily follow differential gene expression patterns that neatly adhere to a canonical M1/M2 phenotype dichotomy.2 It is understood, however, that tumor-associated macrophages typically present an anti-inflammatory or reparatory (M2-like) phenotype that promotes tumor growth, undermines immunotherapeutic response, and correlates with poor patient prognosis.3 Promoting an inflammatory (M1-like) phenotype in tumor-associated macrophages is therefore one approach to inhibit cancer progression.4,5 In contrast, M1-like macrophages initially dominate the immune landscape in injured tissue and participate in the clearance of apoptotic cells and early tissue remodeling.6 A subsequent transition toward populations dominated by an M2-like phenotype is required for tissue healing,7 including by mitigation of inflammatory cytokine production and concurrent growth factor production that is lacking in many contexts and leads to progressive disease (e.g., the development of ischemic heart failure and chronic kidney disease).8,9 The inhibition of inflammatory signaling and modulation of macrophage phenotype has therefore emerged as a critical therapeutic target in tissue healing.10,11

These examples highlight the plasticity of macrophage phenotypes that may be modulated, such as by environmental signals, intercellular mediators, and various drugs for therapeutic benefit.12 Additionally, macrophages are professional phagocytes that ingest and remove damaged cells, foreign materials, and debris from damaged tissues. These functions have been widely exploited for macrophage-targeted drug delivery by nanomaterials.13−15 Applications include phenotypic control in cancer immunotherapy and regenerative medicine. Such targeted delivery approaches have the potential to maximize drug dose at the site of interest while minimizing off-target exposure that is particularly valuable in therapies for cancer, neurological diseases, and immunological conditions.16

Nanoparticles (NPs) are widely used for cell or tissue targeting and therapeutic delivery. NP properties, such as chemical composition, size, shape, and charge, affect how they interact with cells,17,18 which is an important aspect to consider during nanotherapeutic development. Upon introduction to the bloodstream, for example, NPs rapidly develop an adsorbed protein corona that influences the properties of NPs within the body, including their endocytosis,19−21 toxicity,22 and biodistribution,23,24 which potentially leads to undesirable outcomes that include provoking undesirable inflammatory response. However, nanogels, a type of spherical NP formed by cross-linked polymer networks that hold water at a high capacity, can mitigate protein corona formation,25 thereby maintaining the native NP surface and allowing it to interact with cells more efficiently.

Nanogels, including those composed of hyaluronic acid, β-glucan, dextran, and other polysaccharides,26−28 have been leveraged for macrophage-targeted drug delivery and in imaging applications.29−31 However, the capacity of such materials for the retention of small molecule drugs is inherently limited by their rapid diffusive release. To address this issue, we have previously developed cyclodextrin nanoparticles (CDNPs) that enable high drug loading capacity through hydrophobic guest–host interactions. These unique drug delivery vehicles have been used for the macrophage-targeted delivery of small molecule drugs, including for cancer immunotherapy and to assuage inflammation.30,32−37 However, the physiochemical factors affecting the macrophage-targeting capacity of polysaccharide-based nanoparticles and CDNPs, in particular, remain underexplored. Understanding these factors is essential toward further enhancing cell-targeted delivery and developing the next generation of macrophage-targeting NPs.

Here, we investigate aspects of CDNP properties and cell–material interactions that influence macrophage uptake in vitro. Specifically, the CDNP diameter was tailored through alteration in the reactant concentrations used during synthesis to enable the study of size effects independent of changes in particle charge or chemical composition. Cell surface receptors on macrophages, such as the mannose receptor38,39 or scavenger receptors,40,41 are known to be crucial for molecular recognition processes initiating particle uptake. However, their role in the recognition and uptake of polysaccharide materials by macrophages has not yet been scrutinized. Therefore, we explored the hypothesis that these receptor-mediated interactions are a mechanism that contributes to the macrophage uptake of CDNPs. The effects of celastrol, a model anti-inflammatory drug, on inflammatory macrophages were further examined to demonstrate the capacity for CDNPs to improve the cell bioavailability of small molecule therapeutics. Here, we have examined the uptake of polyglucose NPs and found that particle diameter can be a handle to improve macrophage uptake. Furthermore, the interaction between macrophages and such NP systems relies in part on receptor-mediated interactions that may be further exploited to improve cell-targeted therapy.

2. Materials and Methods

2.1. Materials

Solvents and general reagents were purchased from Sigma-Aldrich or TCI and used without additional purification. Cell culture reagents were purchased from VWR unless otherwise stated. Celastrol was purchased from the Cayman Chemical Company.

2.2. Cell Culture

All cells were maintained under standard culture conditions (37 °C, 5% CO2) in the indicated medium. Media was replenished every 2 days, and cells were passaged at 70% confluency. RAW264.7 cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin–streptomycin (Pen-Strep). RAW-Blue (InvivoGen) cells were cultured in DMEM with 10% FBS, 1% Pen-Strep, and 100 μg/mL Normocin. Every other passage, 100 μg/mL Zeocin was added to maintain selection pressure, per manufacturer recommendations. Bone-marrow-derived macrophages (BMDMs) were isolated by standard methods. In brief, bone marrow was extracted from the femur and tibia of male C57BL/6 mice, dissociated, and filtered (40 μm strainer). Red blood cells were lysed prior to cell plating at 2 × 106 cells/well in 24-well plates and maintained in Iscove’s Modified DMEM (IMDM) supplemented with 10% heat-inactivated FBS, 1% Pen-Strep, and M-CSF (10 ng/mL; PeproTech 315-02). Animal procedures were approved by the University’s Institutional Animal Care and Use Committee and performed in compliance with Drexel University’s guidelines for the care and use of laboratory animals. For flow cytometry experiments, RAW264.7 cells or BMDMs were treated with either lipopolysaccharide (LPS; 100 ng/mL) or IL-4 (10 ng/mL; PeproTech 214-14) to polarize cells toward M1-like and M2-like reference phenotypes, respectively.

2.3. Nanoparticle Synthesis and Characterization

Detailed methods for CDNP synthesis have been previously reported.4 Briefly, succinyl-β-cyclodextrin (Thomas Scientific C919U03), 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were dissolved in MES buffer (50 mM, pH 6.0) and stirred for 30 min prior to the dropwise addition of l-lysine and overnight cross-linking. The product was then recovered by precipitation from a 10-fold excess of cold anhydrous ethanol and redissolved in water. Subsequently, the product was purified by size-exclusion chromatography (PD-10, Thermo Fisher 45-000-148), concentrated via centrifugal filtration [10 kDa molecular weight cut-off (MWCO), Thermo Fisher UFC501096], and washed repeatedly with Milli-Q water.

To determine how different sizes of CDNPs affect cell uptake, NPs of varying diameters were synthesized by altering reactant concentrations; the EDC feed ratio was varied (5–12.5 equiv to succinylate groups) while proportionately adjusting the NHS concentration (0.5 equiv to EDC). Particle size was measured using dynamic light scattering (DLS, Zetasizer, Malvern) at a CDNP concentration of 5 mg/mL in 100 mM phosphate-buffered saline (PBS; n = 3). The surface charge of CDNPs was measured using a Zetasizer at a concentration of 100 μg/mL in 10 mM PBS (n = 3). For fluorescence imaging, 10 mg/mL of CDNP was labeled by reaction with 10 μg/mL of Alexa Fluor 555 succinimidyl ester (Thermo Fisher A20009) in carbonate buffer (100 mM, pH 8.5). The reaction proceeded in the dark for 3 h prior to concentration and washing of the final product (CDNP-AF555) by centrifugal filtration with Milli-Q water. Final CDNP products were lyophilized, dissolved in PBS at 10 mg/mL, and stored at −20 °C for later use. To determine the fluorophore label density, absorption was measured at 555 nm (Nanodrop), and fluorophore concentration was calculated using the Beer–Lambert equation (A = γbc), where A is the absorbance, γ is the molar absorptivity (155 000 M–1 cm–1 for AF555), b is the path length, and c is the concentration.

2.5. In Vitro Nanoparticle Uptake

RAW264.7 cells or BMDMs were plated (2 × 104 cells/well) in a 96-well polymer coverslip plate (Ibidi 89626). After overnight culture, cells were treated with 100 μg/mL of CDNP-AF555 at specified time points over 24 h. Cells were then fixed (4% paraformaldehyde, 15 min, 37 °C) and stained for cell membrane [5 μg/mL of Alexa Fluor 488 wheat germ agglutinin (WGA-AF488); Thermo Fisher W11261] and nucleus (NucBlue, Thermo Fisher R37606) prior to imaging (Leica DMI 6000B and Zeiss LSM700). CDNP uptake was analyzed via ImageJ and quantified as the integrated fluorescent density per cell normalized to the cell control after background subtraction.

To investigate receptor-mediated uptake of CDNPs, RAW264.7 cells were plated (2 × 104 cells/well) in a 96-well polymer coverslip plate. Cells were treated with either blocking antibodies for SR-A1 (BD Biosciences, clone U23-56) or MRC1 (Invitrogen, clone MR6F3), each at 20 μg/mL. After 4 h, cells were treated with 100 μg/mL of CDNP-AF555. At 24 h after NP addition, cells were fixed, stained, imaged, and analyzed as previously described.

Flow cytometry was used for further analysis of the receptor-mediated interactions. RAW264.7 cells or BMDMs were plated in 12-well plates (1 × 106 cells/well) polarized to M1-like or M2-like phenotypes, as described above, and subsequently treated with 100 μg/mL of CDNP-AF555. After 24 h, cells were processed for flow cytometry by Fc receptor blocking (BioLegend 101320) and staining with Live/Dead Fixable Aqua (Thermo Fisher L34957) for 15 min at 4 °C in PBS. Cell surfaces were then stained with anti-CD204 (BD Biosciences U23-56) for 30 min at 4 °C prior to washing with flow cytometry staining buffer (Thermo Fisher 2486694) and fixing with Fixation/Permeabilization buffer (Thermo Fisher 88-8824-00) for 30 min at 4 °C. For intracellular staining, cells were stained with anti-CD206 (Thermo Fisher, clone MR6F3) for 1 h in permeabilization buffer. After washing twice with permeabilization buffer, cells were scraped and transferred to 5 mL polystyrene round-bottom tubes (Corning 352054) in flow cytometry staining buffer and run on a BD LSR Fortessa. Data was analyzed using FlowJo v10.9.0. The number of events recorded for all samples ranged from 50 000 to 100 000.

2.7. In Vitro Drug Treatment

To assess the capacity for CDNPs to improve drug bioavailability, we investigated the in vitro delivery of celastrol (a model anti-inflammatory drug). Celastrol was loaded into CDNPs (10 mg/mL) by overnight mixing in water (Cel-CDNP). RAW-Blue cells were plated in 96-well plates (25 × 103 cells/well). Cells were activated using zymosan (100 μg/mL; Thermo Fisher Z2849), a TLR2 agonist, and concurrently treated with free drug or drug-loaded CDNPs of equivalent dose (1 mM to 1 μM celastrol in log-fold dilution; 100 μg/mL of CDNP). After overnight incubation, inflammatory activity was determined using QUANTI-Blue Solution (InvivoGen rep-qbs), following the manufacturer’s protocol.

2.6. Statistical Analysis

Data are presented as the mean ± standard deviation (SD) unless otherwise stated. Statistical analysis was performed using GraphPad Prism v10.0.0 using analysis of variance (ANOVA) with post hoc Tukey’s honestly significant difference (HSD) test or unpaired t-test where appropriate. Significance was determined at P < 0.05.

3. Results and Discussion

3.1. CDNP Uptake Is Dependent on Particle Diameter

NP uptake is heavily influenced by a multitude of physiochemical parameters that include shape, charge, surface coatings, and size.18 Surface chemistry can likewise be used to exploit particle–receptor interactions to facilitate specific uptake mechanisms;42,43 in many cases, the formation of the protein corona and related surface labeling by opsonins contributes to phagocytic clearance by macrophages.44,45 For example, the role of surface charge on polystyrene NP uptake has been investigated, finding that charge positively correlated with uptake.46 In another study, NP surface modification by CD47 (a “don’t eat me” signal47) decreased macrophage uptake, specifically in M1-like macrophages.48 These interactions between cells and nanomaterials can thus be exploited to improve macrophage-targeted drug delivery by NPs, including polysaccharide-based nanoparticles.

β-Cyclodextrin (CD) is a polysaccharide and macrocyclic host composed of seven d-glucose units arranged in a toroidal fashion through α-1,4-glycosidic bonds. It readily forms physical associations (i.e., guest–host complexes) with appropriately sized hydrophobic guest molecules, including a diverse array of small molecule drugs. This property is widely used in the pharmaceutical industry to enhance drug pharmacokinetics via improved solubility and bioavailability.49 Here, cyclodextrin nanoparticles (CDNPs) were prepared via cross-linking of succinyl-β-CD with l-lysine mediated by EDC/NHS (Figure 1a). CDNPs formed by these methods are spherical nanogels with a moderate negative charge and a dense concentration of host sites to allow for high drug loading. CDNP uptake by myeloid cells (e.g., macrophages and dendritic cells) has been previously used for the targeted delivery of TLR7/8 agonists,4,30 NF-κB inhibitors,33 and antiarrhythmic drugs.32 Notably, treatment by CDNPs alone does not affect cell viability or macrophage polarization.4,37 Here, we sought to determine how CDNP size affects their uptake by macrophages. We previously demonstrated that altering EDC feed ratio (i.e., the reactant concentration relative to succinyl groups) provides CDNPs of varying diameters,4,37 affording a broader range of particle diameters than by varying CD or l-lysine concentrations. Moreover, these latter methods also altered the final chemical composition, which is unaffected by the alteration of EDC and NHS concentrations. It is expected that increased particle diameter is attributable to improved cross-linking kinetics afforded by overcoming hydrolysis of EDC and the requisite succinimidyl ester intermediate. Here, we have varied the EDC feed ratio from 5:1 to 12.5:1 to generate CDNPs that range in diameter from 23.92 ± 0.66 to 123.27 ± 4.34 nm (Figure 1b). Importantly, because l-lysine and CD substrate concentration remain unchanged, the surface charge of all CDNPs formed was similar across groups (Figure S1).

Figure 1.

Figure 1

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CDNP synthesis and characterization. (a) Schematic of cyclodextrin nanoparticle (CDNP) preparation via EDC/NHS-catalyzed cross-linking of succinyl-β-cyclodextrin by l-lysine and subsequent fluorescence derivatization by reaction with the succinimidyl ester of Alexa Fluor 555. (b) The dependence of CDNP on the molar feed ratio of EDC to succinyl groups (3.3% w/v cyclodextrin, 0.5:1 l-lysine), n = 3. (c) Absorbance (solid line) and emission (dashed line) spectra of CDNP-AF555 in PBS.

RAW264.7 cells were treated with fluorescently labeled CDNPs (CDNP-AF555) of varying sizes. Importantly, wavelengths for absorbance and emission were relatively unaffected by labeling of CDNPs with AF555 (Figure 1c), and the degree of labeling was consistent across all groups, which ranged from 80 to 120 μmol dye/mg of CDNP (P = 0.85, ANOVA; mean of 98.06 ± 16.28 μmol dye/mg across all groups). NP uptake was monitored over 24 h (Figure 2a), during which time uptake was observed across the cell population after treatment with CDNPs of varying diameter. Cell segmentation and quantification of uptake revealed that increasing CDNP size generally led to a greater extent of NP uptake over time (Figure 2b), which was highly significant at the 24 h time point (Figure 2c). These observations are consistent with literature reports that larger nanomaterials are more efficiently taken up by phagocytes, including macrophages of varying phenotypes.50,51 Our results contribute to the growing literature on the properties of NPs (such as size, shape, and rigidity) that dictate uptake and immune responses in different cell types, specifically by showing that polyglucose nanogel uptake is positively correlated with increased NP size.

Figure 2.

Figure 2

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Time-course imaging of CDNP uptake in RAW264.7 macrophages. (a) Representative fluorescence images of CDNP-AF555 (12.5:1 equiv of EDC) uptake at 0, 6, and 24 h. Staining: DAPI (nuclei, blue), WGA-AF488 (cell membrane, green), and CDNP-AF555 (nanoparticle, red). Scale bars: 50 μm, primary; 10 μm, inset. (b) Quantification of CDNP-AF555 uptake over time and dependence on varying CDNP diameter over 24 h normalized to untreated controls; mean ± SEM, n > 100 cells. (c) Integrated density at 24 h normalized to untreated cell controls; mean ± SD, n > 100 cells; ****P < 0.0001; ANOVA, Tukey’s HSD.

3.2. Nanogel Uptake Is Dependent on Receptor-Mediated Interactions

To better understand the mechanisms underlying nanogel uptake, we next investigated how this was impacted by macrophage phenotype and specific receptor-mediated interactions. Although the understanding of macrophage phenotypes and the associated nomenclature is continually evolving,52 we here use standard reference phenotypes that include untreated (M0) controls, M1-like (inflammatory, LPS treated), and M2-like (reparatory, IL-4 treated). Subsequent to polarization, the cells were treated with CDNP-AF555 and examined via flow cytometry. In these studies and the following, cells were treated with the largest size CDNP (12.5:1 equiv) to further understand the mechanisms that contribute to uptake of the most rapidly internalized material composition. The mean fluorescence intensity (MFI) was generally similar across treatment groups but with a significant increase for the M1-like phenotype (Figure 3a,b). A relative increase in the frequency of CDNP+ cells was likewise observed for M1-like populations (Figure 3c). Interestingly, bone-marrow-derived macrophages (BMDMs) exhibited a notably different uptake response with enhanced uptake by the M2-like phenotype (Figure S2). Targeted delivery of NPs to specific macrophage phenotypes customarily leverages the surface conjugation of ligands for receptors that exhibit phenotype-dependent expression patterns, such as mannose or folate receptors.53−55 We hypothesized that a greater expression level of receptors involved in uptake may be driving the observed polarization-dependent differences in CDNP internalization. Moreover, the variability of receptor expression within these populations may underlie the heterogeneous uptake observed in single-cell image analysis (Figure 2c).

Figure 3.

Figure 3

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Flow cytometry of CDNP uptake by RAW264.7 macrophages. (a) Representative histograms and (b) corresponding quantification of CDNP-AF555 mean fluorescence intensity (MFI) for M0, M1, and M2 polarized cells with and without CDNP treatment. (c) Quantified frequency of CDNP+ cells. Data represents the mean ± SD, n = 3; ****P < 0.0001; ANOVA, Tukey’s HSD.

To further understand cell–material interactions that may be later exploited to improve the efficacy of delivery, direct molecular recognition via receptors and other modes of NP internalization are important facets to consider. While a number of pattern recognition receptors (PRRs) may contribute to the recognition and uptake of foreign materials, we hypothesized that scavenger receptor (e.g., SR-A1) and mannose receptor (MRC1) are involved in the CDNP–macrophage interaction. We specifically investigated these receptors because scavenger receptors are critical in the recognition of negatively charged materials,56 such as our succinyl-containing CDNPs, and mannose receptors bind to a variety of oligosaccharides, including not only mannose but also galactose, glucose,39 and potentially our polyglucose-based NPs. In both RAW264.7 cells and BMDMs, the expression of SR-A1 (CD204) and mannose receptor (CD206) was dependent upon the cell polarization state (Figure S3). To investigate the putative receptor–particle interactions driving macrophage uptake, we used flow cytometry to examine the receptor expression levels of CD204 and CD206 in M0, M1-like, and M2-like macrophages in relation to CDNP uptake (Figure 4a, Figure S4a). Flow cytometry plots indicated a propensity for increased receptor expression to be associated with a greater extent of CDNP uptake. Furthermore, a greater abundance of CDNP+CD204hi and CDNP+CD206hi cells was present in M1-like RAW264.7 cells and M2-like BMDMs (Figure 4b,c, Figure S4b,c, and Figure S5), which is indicative of the receptors’ role in polyglucose NP uptake and in driving the phenotype-dependent differences in uptake. Interestingly, we likewise noted an increase in CD204 and CD206 expression levels in response to CDNP treatment; this phenomenon could be beneficial for macrophage-targeted drug delivery if similar results are later validated in vivo.

Figure 4.

Figure 4

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Flow cytometry of receptor expression and CDNP uptake in RAW264.7 macrophages. (a) Representative flow cytometry plots for RAW264.7 macrophages with or without CDNP-AF555 treatment. Gating represents regions of CDNP+ uptake coincident with CD204hi (left) or CD206hi (right) expression. Inset values represent percentage of the live population that is (b) CDNP+CD204hi or (c) CDNP+CD206hi. Data represents the mean ± SD, n = 3; ****P < 0.0001; ANOVA, Tukey’s HSD.

Antibody blockade of SR-A1 and MRC1 was used to validate these observations. Blocking of MRC1 and SR-A1 in unpolarized RAW264.7 cells reduced CDNP uptake, each to a different degree (Figure 5a,b), which verifies that both receptors contribute to NP uptake. However, CDNP uptake was not completely diminished by blocking these receptors, indicating that other mechanisms must be involved in their internalization. Phagocytosis is often regarded as the primary mechanism of uptake by macrophages,57 in particular for small particles (<6 μm).58 However, other mechanisms, including pinocytosis,59,60 micropinocytosis, and clathrin-mediated endocytosis, likewise contribute to the internalization of nanomaterials (fluids, 0.5–5 μm, and 20–500 nm, respectively).61−63 Cytochalasin D (CytoD), an inhibitor of actin polymerization, has been used previously to block phagocytosis and pinocytosis in many cell types.64,65 RAW264.7 cells were first treated with CytoD to inhibit these mechanisms and then treated with CDNP to monitor uptake. CytoD treatment reduced CDNP uptake to a greater degree than receptor inhibition, indicating that these nonspecific uptake routes further contribute to CDNP uptake in vitro (Figure 5a,b). Importantly, treatment with blocking antibodies and CytoD did not affect the metabolic activity of the cells that is indicative of viability (Figure S6); results are therefore attributable to the specific putative blocking mechanisms involved in NP internalization. In sum, results here demonstrate that general mechanisms of macrophage pinocytosis contribute to CDNP internalization, similar to innumerous drug delivery systems.66 Receptor-mediated endocytosis, however, contributes to the uptake of polyglucose nanomaterials by macrophages and is likely a critical mechanism of cell-targeted delivery that may be critical in vivo, where particulate concentrations are too low for pinocytosis alone to be an efficient targeting mechanism. While the results do not rule out the contribution of other specific receptors, they implicate the involvement of CD204 and CD206 in the process of polyglucose NP uptake.

Figure 5.

Figure 5

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Cell imaging for CDNP uptake inhibition. (a) Representative fluorescence images of CDNP-AF555 uptake in RAW264.7 macrophages at 24 h after indicated antibody blocking (MRC-1, SR-A1) or CytoD treatment. Staining: DAPI (nuclei, blue), WGA-AF488 (cell membrane, green), and CDNP-AF555 (nanoparticle, red). Scale bar: 50 μm. (b) Corresponding quantification of CDNP-AF555 uptake normalized to CDNP-AF555 treated controls; mean ± SEM, n > 100; **P < 0.01, ****P < 0.0001; ANOVA, Tukey’s HSD.

3.3. Cyclodextrin Nanoparticles Aid Drug Delivery to Macrophages

Nanotherapeutic drug delivery vehicles are often intended to improve the bioavailability of their drug cargo, including through active transport into the target cells. To demonstrate the capacity for CDNPs to serve as such a drug vehicle, we explored the activity of anti-inflammatory drug-loaded CDNPs. Celastrol has been previously identified as a potent immunomodulatory therapeutic, which inhibits M1-like activation (IC50 < 100 nM for NF-κB inhibition) and promotes M2-like polarization both in primary murine and human cells.67,68 The effect of celastrol-loaded CDNPs was examined in RAW-Blue reporter cells that incorporate a SEAP reporter gene downstream of NF-κB and AP-1 promoter regions to allow direct assessment of inflammatory activation. Cells were stimulated by zymosan, a TLR2 agonist that mimics sterile inflammation,37,69 and concurrently treated with drug-loaded CDNPs or soluble celastrol. Importantly, celastrol can be encapsulated in CDNPs with simple mixing in water without the need for additional purification steps. Thus, the desired drug dose is consistent across all treatment groups. Celastrol-loaded CDNPs (Cel-CDNP) suppressed inflammatory signaling by greater than 60% and in a dose-dependent fashion (Figure 6a). Further, when comparing the anti-inflammatory capacity between soluble celastrol (1 μM) and Cel-CDNP, nanotherapeutic-assisted delivery improved outcomes at most CDNP sizes (Figure 6b). While larger CDNPs may be anticipated to best improve drug efficacy because of their more rapid internalization, results were relatively independent of particle size. This finding may be attributable to alteration in drug release kinetics or other factors. While in vitro studies fail to account for critical alterations in drug pharmacokinetics (e.g., increased blood half-life, alterations in biodistribution) that may occur in vivo, these findings corroborate the suitability of CDNPs as a carrier for macrophage-targeted drug delivery that enhances drug bioavailability at the cellular level.

Figure 6.

Figure 6

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Bioavailability of celastrol-loaded CDNP (Cel-CDNP). (a) Anti-inflammatory activity of Cel-CDNP treatment on RAW-Blue cells assessed 24 h after concurrent drug treatment and zymosan stimulation; mean ± SD, n = 3. (b) Anti-inflammatory activity of unloaded CDNP vehicle, free celastrol (1 μM), and Cel-CDNP of varying sizes; mean ± SD, n = 3; −P < 0.05 relative to zymosan (positive) control; *P < 0.05 relative to free celastrol treatment; ANOVA, Tukey’s HSD.

4. Conclusions

Macrophages are critical regulators of the immune microenvironment, phenotypic modulation of which is a crucial therapeutic target. Polyglucose materials, including those formed from dextran and cyclodextrin, are known to exhibit a high macrophage avidity that has been used for image-based diagnostics and drug delivery applications. Here, we have tuned intermediate reactant concentrations used during the synthesis of cyclodextrin-based nanogels (rather than substrate concentrations) to vary particle size without altering their chemical composition or the corresponding surface charge. Size positively influenced the extent of nanogel uptake, and image analysis revealed a high degree of heterogeneity across the cell populations. Preferential uptake was likewise dependent on macrophage polarization and the cell source. Both heterogeneity of uptake at the single-cell level and cell-state dependent differences are likely attributable to the differential expression of pattern recognition receptors (specifically, scavenger receptor A1 and mannose receptor) by these cell types. While nanogel-assisted delivery moderately improved the bioavailability and therapeutic effects of an anti-inflammatory drug in vitro, these effects may be more profound in vivo where issues of drug biodistribution and rapid renal clearance further contribute to the poor bioavailability of small molecule therapeutics. Overall, CDNPs are a promising macrophage-targeted drug delivery vehicle, the application of which warrants continued study. The insights provided by this work add to the current understanding of cell–material interactions that drive macrophage avidity for polysaccharide-based materials and inform the continued development of nanogels for the phenotypic control of macrophages that is under exploration for cancer immunotherapy and immunoregenerative medicine.

Acknowledgments

This work was supported by Startup Funds from the School of Biomedical Engineering, Science and Health Systems at Drexel University, NIH R35GM147184, and an American Heart Association Career Development Award (10.58275/AHA.23CDA1054367.pc.gr.168006).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsabm.3c00985.

  • Flow cytometry quantification of CDNP+ cell populations in M0, M1, and M2 polarized BMDMs; flow cytometry plots and corresponding quantification of receptor expression and CDNP uptake in BMDMs; and time-course imaging of CDNP uptake by BMDMs with quantification (PDF)

Author Contributions

S.S.S. and C.B.R. developed the materials concept and experimental design. S.S.S. and K.M.K. performed the material synthesis, characterization, and data analysis. All authors contributed to interpretation of results and preparation of the manuscript with approval of the final version of the manuscript.

The authors declare the following competing financial interest(s): C.B.R. is listed on a patent filed by Partners Healthcare pertaining to the development of the CDNP. The remaining authors declare no competing interests.

Supplementary Material

mt3c00985_si_001.pdf (703.5KB, pdf)

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