Drug-loaded nanoparticles induce immunogenic cell death and efficiently target cells from glioblastoma patients

ABSTRACT

Aim

Glioblastoma multiforme (GBM) is characterized by a highly immunosuppressive tumor microenvironment (TME), posing significant challenges for efficient therapy’s outcomes. Nanomedicine combined with immunotherapy holds the potential to modulate the TME and reactivate immune responses. This study proposes a polymeric nanosystem (NPs) encapsulating diaminocyclohexane-platinum II (DACHPt), an oxaliplatin derivative, to induce immunogenic cell death (ICD) in GBM cells.

Materials & methods

An ionic-gelation technique was employed to generate polymeric nanoparticles (NPs) with an approximate size of 200 nm. NPs internalization was analyzed in GBM cell lines, in vitro-derived macrophages, and in leukocytes and tumor cells from GBM patient via flow cytometry and confocal imaging. ICD was assessed by measuring two of its main markers: adenosine triphosphate (ATP) and high-mobility group box 1 (HMGB1).

Results

NPs were efficiently incorporated by myeloid and tumor cells, but not by lymphocytes. DACHPt-loaded NPs demonstrated enhanced cytotoxicity compared to free drug, with increased ATP and HMGB1 release from GBM cells, confirming ICD induction.

Conclusions

Our findings suggest that DACHPt-loaded NPs represent a promising therapeutic strategy capable of targeting both tumor cells and tumor-promoting immune cells while inducing ICD.

1. Introduction

The tumor microenvironment (TME) is a dynamic ecosystem comprising a diverse array of cellular and non-cellular components, including tumor cells, immune cells, and the extracellular matrix (ECM) [1]. This intricate interplay significantly influences tumor progression, metastasis, and response to therapy. In recent years, the critical role of immune cells within the TME has emerged, particularly in the context of cancer immunotherapy [2,3].

Glioblastoma multiforme (GBM), an aggressive primary brain tumor, exemplifies the complex crosstalk between tumor cells and the immune system. Characterized by its immunosuppressive microenvironment, GBM is often referred to as a cold tumor due to the limited infiltration of T cells. This immunosuppressive state, orchestrated by various immunosuppressive mechanisms, hinders effective anti-tumor immunity and contributes to poor patient outcomes [4,5].

Despite significant advancements in cancer therapy, including immunotherapy, the prognosis for GBM patients remains dismal [6–8]. While targeting specific immune cell subsets within the TME holds promise, the unique challenges posed by the brain’s immune privilege and the complex nature of the GBM TME necessitate a deeper understanding of this intricate ecosystem [9].

To overcome these challenges, innovative therapeutic strategies are needed to modulate the GBM TME and stimulate anti-tumor immunity. Nanotechnology-based drug delivery systems offer a promising approach to enhance the efficacy of cancer therapies, including immunotherapy [10]. By precisely targeting drug delivery to tumor cells and immune cells, nanomedicines can improve therapeutic outcomes while minimizing systemic toxicity. Specifically, in the context of immunotherapy, nanomedicines have demonstrated significant capacity in enhancing the activation of local immune responses and facilitating the delivery of unstable proteins and genetic material for immune modulation [11–15].

In this study, we investigated the potential of an innovative drug-loaded nanosystem for inducing specific danger signals associated with immunogenic cell death (ICD). Unlike other forms of cell death, such as apoptosis or necrosis, ICD is characterized by the release of distinct molecules known as damage-associated molecular patterns (DAMPs) [16]. These DAMPs have the capacity to stimulate the immune system and generate a robust anti-tumor response. Induction of ICD has already been proposed as an innovative strategy to stimulate the antitumor immune response in cold tumors like GBM [17]. The drug encapsulated within our nanosystem, diaminocyclohexane-platinum II (DACHPt) is a potent ICD inducer [16,18], where its chemical structure makes it more stable and compatible with polymeric carriers, enhancing its applicability in advanced drug delivery platforms [19–23].

The aim of this work was to demonstrate that a DACHPt-loaded nanosystem can function as an effective strategy to convert a “cold” TME, such as that found in GBM, into a more immunologically active “hot” environment through ICD induction and the depletion of tumor-promoting cells, while minimizing systemic side effects.

2. Materials & methods

2.1. Patient characteristics

Patients were recruited at the Department of Neurosurgery, Padova University Hospital and Florence University Hospitals (from 2021 to 2023). All the experiments were approved by the ethics committees of the Veneto Institute of Oncology – IRCCS of Padova, Italy (MDSC_SNC 2016/13) and the Padova and Florence University Hospitals (NOI_NCH 1536/19). All patients gave their written informed consent, and the study was conducted in accordance with the Declaration of Helsinki. All patients included in this study had a confirmed diagnosis of glioblastoma (GBM, glioma grade 4, IDH1 WT – as defined by the 2021 WHO classification) by standard histopathological and molecular analyses. In all cases 5-ALA-assisted surgery was employed, tissue specimens were derived from the protoporphyrin IX (PPIX) bright fluorescent area, corresponding to the central non-necrotic area [24].

2.2. Tumor and blood sample processing

Tumor samples were either processed on the same day of resection or kept at 4°C in MACS Tissue Storage Solution (Miltenyi Biotec, Bergisch Gladbach, Germany) and processed the following day as previously described [24]. Tissue specimens were washed with 0.9% sodium chloride to remove peripheral blood traces and then digested mechanically and enzymatically by using Tumor Dissociation Kit (Miltenyi Biotec) and gentle MACS Octo Dissociator (Miltenyi Biotec) according to the manufacturer’s instructions for soft tumors to obtain a single cell suspension. Peripheral blood was drawn from patients either at surgery before anesthesia induction, or the day before surgery, and processed immediately or the following day.

2.3. Chemicals

Dichloro (1,2-diaminocyclohexane) platinum (II) (DACHPt-Cl2, Mw = 380.17 Da), AgNO3 (Mw = 169.97 Da), mannitol (Mw = 182 Da), N1 medium supplement 100X (0.5 mg/ml recombinant human insulin, 0.5 mg/ml, human transferrin – partially iron-saturated-, 0.5 μg/ml sodium selenite, 1.6 mg/ml putrescine, and 0.73 μg/ml progesterone) and Dulbecco’s Phosphate Buffer Saline (PBS) were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Poly-L-Arginine (PA-Cl) hydrochloride (Mw = 5800 Da) was purchased from Alamanda® Polymers (Huntsville, Alabama, USA). Low molecular weight HA (Mw  ≈  20 kDa) was purchased from Lifecore® Biomedical (Chaska, Minnesota, USA). MTS CellTiter 96® AQueous One Solution cell proliferation assay kit, RealTime-Glo™ Extracellular ATP Assay and Lumit™ HMGB1 (Human/Mouse) Immunoassay were provided by Promega (Charbonnières-les-Bains, France). Deionized water (MilliQ water) was obtained from a Milli-Q plus system (Merck-Millipore, Darmstadt, Germany). Oxaliplatin solution was kindly provided by UOC Pharmacy Veneto Institute of Oncology (IOV-IRCCS). Fluorochrome Cyanine 5 was purchased from Lumiprobe.

2.4. Preparation and physico‑chemical characterization of polymeric nanoparticles

Blank, fluorescent and DACHPt-loaded hyaluronic acid (HA)-polyarginine (PA) nanoparticles (NPs) were synthesized using the ionic gelation technique. The technique relies on electrostatic interactions between oppositely charged polymers, such as positively charged polyarginine and negatively charged hyaluronic acid. The key mechanism for DACHPt association with nanoparticles is the formation of a metal-polymer complex between platinum and the carboxyl groups of HA [21]. Cy5-labeled NPs were produced by substituting 30% w/w of HA with Cy5-HA synthesized based on method described by Andretto et al. 28 and Fudala et al. [25,26]. Size, estimated by the average hydrodynamic diameter, polydispersity index (PDI) and zeta (ζ)-potentials were determined by dynamic light scattering (DLS) using NanoZS® (Malvern Instruments, Worcestershire, United Kingdom). Transmission electron microscopy (TEM) was conducted using a Philips CM120 microscope at the “Centre Technologique des Microstructures” (CTμ) of the University of Lyon 1 (Villeurbanne, France). A 10 μL drop of the suspension was placed on a carbon/formvar microscope grid (Delta Microscopies, Saint-Ybars, France), stained with a 1% w/w aqueous solution of sodium silicotungstate (Agar Scientific, Rotherham, UK), and allowed to dry slowly in open air. The dried samples were then observed by TEM at an acceleration voltage of 120 kV.

2.5. Cells

In vitro differentiation of macrophages from blood monocytes was carried out as previously described [27]. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors using a density gradient centrifugation on Ficoll-Paque PLUS (GE Healthcare-Amersham, Buckinghamshire, UK). Monocytes were separated from lymphocytes by adhesion and cultured in 24-well plates for 7 days with 100 ng/ml M-CSF (MiltenyiBiotec). U87MG and A172 cell lines were purchased from the American Type Culture Collection (ATCC). U87MG is a hypodiploid human cell line that was isolated from malignant gliomas from a male patient with glioblastoma. A172 cell line has a karyotype of 80 chromosomes and originates from the brain tissue of a male patient with glioblastoma. Both cell lines grow in adherence using the medium Dulbecco’s Modified Eagle Medium, DMEM high glucose (EuroClone) supplemented with 10% FBS, 100 U/ml penicillin/streptomycin, 10 mM Hepes, 0.67 mM arginine, 0.28 mM asparagine, 1.5 mM glutamine. Cells were confirmed mycoplasma-free by testing with the LookOut® Mycoplasma PCR Detection Kit (Sigma-Aldrich).

2.6. NPs incorporation

GBM cell lines, in vitro-derived macrophages, blood leukocytes and tumor cell suspension obtained as previously described were incubated with fluorescent NPs at the final polymers’ concentration of 50 μg/ml for 3 h. As control, Blank NPs formulations were used. At the end of the incubation, cells were stained for flow cytometry analysis and confocal imaging studies.

2.7. Multiparametric flow cytometry

Single-cell suspensions from GBM tissue were counted and stained with LIVE/DEAD Fixable Aqua (Life Technologies, Thermo Fisher Scientific, Waltham, Massachusetts), anti-CD45 BV421 (BD Biosciences, Becton Dickinson, Franklin Lakes, New Jersey, USA), anti-CD33 PE-Cy7 (eBioscience, Thermo Fisher Scientific, Waltham, Massachusetts, USA), anti-HLA-DR FITC (BD Biosciences) and anti-CD49d PE (BioLegend, San Diego, California, USA). Also, blood leukocytes were counted and stained with LIVE/DEAD Fixable Aqua (Life Technologies, Thermo Fisher Scientific, Waltham, Massachusetts, USA), CD14-FITC (BD Biosciences), CD15-V450 (BD Biosciences), CD3-PE-Cy7 (Beckman Coulter, Indianapolis, Indiana, USA). Data acquisition was performed using LSRII flow cytometer (BD Biosciences) and results were analyzed by FlowJo software (BD Biosciences).

2.8. Confocal studies

U87MG cells and in vitro-derived macrophages were seeded on coverslips in 24-well plates for 2 h and then incubated with fluorescent NPs and prepared for confocal analysis. Cells were fixed with 4% (v/v) paraformaldehyde in PBS, for 15 min at room temperature. Macrophages were stained with CD33-FITC (BD Biosciences) and U87MG were stained with CD44-FITC (Beckman Coulter) (both dilutions 1:200). Subsequently, cells were stained with DAPI (stock at 20 mM diluted 1:2000) for 1 h at room temperature. Samples were finally mounted in ProLong Diamond Antifade Mountant (Invitrogen). Slides were analyzed under a Zeiss LSM900 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany) using a 40× or a 63× objective (Plan-Apochromat 63 × /1.40 oil DIC M27). For fluorescence excitation, a diode laser at 405 nm for DAPI, a diode laser at 488 nm for Alexa fluor 488 and a diode laser at 640 nm for Cyanine 5 were used. The images were processed using ZEN 3.2 Blue Edition software (Zeiss).

2.9. Viability studies on U87MG and A172 cell lines and in vitro-derived macrophages

U87MG cells (1 × 10⁴ per well), A172 cells (1 × 10⁴ per well) and macrophages (7 × 10⁴ per well) were plated into 96-plates and then treated for 24 hours with increasing concentrations of blank NPs, DACHPt-NPs and oxaliplatin solution. Cell viability reduction assay was performed using CellTiter 96® AQueous One Solution cell proliferation assay kit (Promega) containing a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2 h-tetrazolium, inner salt; MTS]. Absorbance at 490 nm (1 sec) was recorded using 2030 Multilabel Reader Victor™ X4 (PerkinElmer) after 1, 2 and 3 hours of incubation at 37°C 5% CO₂ with 120 μl of 1:6 diluted MTS solution. Cell viability (CV) percentage was evaluated through the following formula, with absorbance control well, the absorbance value of untreated cells. To minimize the interference of cell culture medium absorbance with the measurements, its absorbance value was subtracted from all samples.

$$\mathit{CV}\left(\mathrm{\%}\right)=\frac{\mathrm{Absorbance}\mathrm{treated}\mathrm{cells}}{\mathrm{Absorbance}\mathrm{untreated}\mathrm{cells}}\mathit{x}100$$

2.10. Extracellular ATP release assay

1×10⁴ U87MG cells per well were plated in a 96 wells-plate and treated with increasing concentrations of DACHPt-NPs, Blank NPs and free oxaliplatin in DMEM complete medium. The selected increasing dosages for each of the three treatments were 12,5 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml, and 200 μg/ml. The release of extracellular ATP was assessed by RealTime-Glo™ Extracellular ATP Assay (Promega) used according to the manufacturer’s instructions. Since ATP is the limiting factor in the reaction, the luminescence is directly proportional to the concentration of ATP present: higher ATP concentration results in a stronger luminescence signal, indicating higher levels of cell death. The bioluminescence was measured using the VICTOR® Nivo™ (PerkinElmer) at various time points, up to the first 8 hours after the treatments were added to the wells. The response index was then calculated using the following formula, where RLU stands for relative light units:

$$\mathit{Release}\mathit{response}\mathit{index}=\frac{\mathrm{Average}\mathrm{RLU}\mathrm{of}\mathrm{replicates}\mathrm{of}\mathrm{treated}\mathrm{cells}}{\mathrm{Average}\mathrm{RLU}\mathrm{of}\mathrm{replicates}\mathrm{of}\mathrm{untreated}\mathrm{cells}}\mathit{x}100$$

2.11. HMGB1 release secretion assay

The secretion of HMGB1 was evaluated by The Lumit™ HMGB1 (Human/Mouse) Immunoassay (Promega) used according to the manufacturer’s instructions. U87MG cells were prepared as indicated for ATP assay. Data analysis was conducted according to protocol guidelines.

2.12. Statistical analysis

Student t-test was used to evaluate statistically significant variations between two groups of samples and one or two-way ANOVA was used to compare three or more groups according to the number of variables. In the ATP release experiment, a Linear Mixed Effects Model was used to compare the effects of the three different treatments using two different concentrations at specific time points. IC50 values were calculated using the model: log (inhibitor) vs normalized response (variable slope) that best fit our data after logarithmic transformation of the drug concentration data. Differences were considered statistically significant with p value ≤ 0.05. All statistical analyses were performed with the GraphPad Prism software version 8 (GraphPad Software, Inc. by Dotmatics).

3. Results

3.1. Characteristics of polymeric nanoparticles and internalization studies

In this study, we employed a polymeric nanosystem comprised of two distinct polymers: hyaluronic acid (HA) and polyarginine (PA). These components were selected based on their established non-toxicity, biodegradability, and biocompatibility profile as previously outlined by Matha et al [21]. In this nanosystem, we encapsulated DACHPt, an oxaliplatin derivate and for internalization studies we used HA labeled with Cyanine 5 (Cy5) to obtain fluorescent nanoparticles. Physicochemical analysis showed that DACHPt-loaded nanoparticles had a hydrodynamic size of around 200 nm with a negative zeta potential ( −30 mV) and encapsulation efficacy (EE) around 70% [21] (Table 1). As showed by TEM images, nanoparticles presented a spherical shape and formed homogeneous distribution (Figure 1).

Table 1.

Physicochemical properties of the empty nanosystem (blank-NPs), fluorescent nanosystem (Cy5-NPs) and DACHPt-NPs.

NPs formulation	Size (nm)	PdI	ζ potential (mV)	pH	EE%	[NP] (mg/ml)	[DACHPt] (mg/ml)
Blank-NPs	206  ±  6	<0.2	−  33  ±  3	5.5	–	1.9	–
Cy5-NPs	184  ±  0.3	<0.2	−  34  ±  3	5.5	–	1.9	–
DACHPt-NPs	161  ±  4	<0.2	−  42  ±  4	5.5	70  ±  3	1.9	0.250

Figure 1.

TEM images of DACHPt-loaded nanoparticles (DACHPt-NPs). Scale bars are 500 nm and 200 nm. Operating voltage = 120 kV.

Initially, to prove the ability of NPs to be incorporated by cells present in the TME, we used the GBM cell line U87MG and in vitro-derived macrophages obtained after differentiation of monocytes isolated from healthy donors. Such in vitro-derived macrophages share several characteristics with tumor-associated macrophages, including the relevant immunosuppressive activity, as previously described [27].

To study the internalization of fluorescent nanoparticles, cells were treated with 50 µg/ml of Cy5-NPs for 3 hours. Following incubation, cells were analyzed by multiparametric flow cytometry and confocal fluorescence microscopy (CFM). Both analytical methods confirmed the presence of the fluorophore Cy5 within all the cells, thus confirming the successful internalization of these nanoparticles (Figure 2). Treatment with empty NPs (Blank-NPs) was also carried out in order to confirm that the positive signal was given only by the incorporation of the NPs and the fluorophore itself, but not due to any increase in autofluorescence after uptake. Moreover, we observed that the incorporation of these NPs is both time and dose dependent, and within two hours maximal uptake was reached (data not shown).

Figure 2.

Evaluation of nanoparticles incorporation by flow cytometry and confocal microscopy in U87MG cells (a) and in vitro-derived macrophages (b). Cells were treated with cyanine 5 nanoparticles (Cy5-NPs) for 3 hours. Representative flow cytometry analysis and confocal images of cells stained with anti-CD44 (U87MG) and anti-CD33 (macrophages) and nuclear dye DAPI, showing the incorporation of fluorescent nanoparticles. Images are reported at a magnification of 63X. Cell size is reported by scale bar (20 µm).

3.2. Evaluation of cell viability following polymeric nanosystem administration

To assess cell viability following NPs incorporation, we performed an MTS assay, and calculated IC₅₀ for each condition. Cell viability assays were performed on glioma cell lines U87MG, A172 and in vitro-derived immunosuppressive macrophages (Figure 3). To this aim, cells were treated for 24 hours with both blank and DACHPt-loaded NPs at the same polymer concentration. DACHPt-NPs were evaluated at drug concentrations ranging from 1 μg/ml to 100 μg/ml. In contrast, oxaliplatin was tested at higher concentrations to achieve maximum cytotoxicity, specifically up to 800 μg/ml for U87MG cells, 600 μg/ml for A172 cells, and 400 μg/ml for in vitro-derived macrophages.

Figure 3.

In vitro cytotoxicity evaluation of nanoparticles on U87MG cells (a), A172 cells (b) and in vitro-derived macrophages (c). Cell viability was evaluated by MTS assays following increasing concentrations of empty nanoparticles (blank-NPs), drug-loaded nanoparticles (DACHPt-NPs) and free oxaliplatin after 24 hours of incubation. Data were normalized assuming 100% viability of untreated cells. Mean ± SD (n = 3).

Results indicate that DACHPt-NPs effectively reduced cell proliferation across all three cells types tested. Blank-NPs and DACHPt-NPs are compared based on the polymer concentration; DACHPt-NPs and oxaliplatin are compared based on the drug concentration. For U87MG and A172 cells, the IC₅₀ value of encapsulated DACHPt was about ten times lower than that of oxaliplatin solution (Table 2). In the case of in vitro-derived macrophages (Figure 3(c)), it was about fourfold lower than free oxaliplatin. Blank-NPs also affected cell viability, but at polymer concentrations at least four times higher than the IC₅₀ value of the encapsulated DACHPt for U87MG and in vitro –derived macrophages, and two times higher for A172 cells (Table 2).

Table 2.

IC₅₀ values of the DACHPt-loaded NPs (DACHPt-NPs) and free oxaliplatin in the three cell types: U87MG and A172 glioma cell lines and in vitro-derived macrophages.

Cell type	Concentration type	Blank-NPs (μg/ml)	DACHPt-NPs (μg/ml)	Oxaliplatin (μg/ml)
U87MG	Drug	–	28.7	290.5
	Polymers	968.3	218	–
A172	Drug	–	26.6	295.4
	Polymers	415.1	202.3	–
Macrophages	Drug	–	15.22	65.8
	Polymers	449.46	115.8	–

3.3. Evaluation of ICD-related DAMPs release in GBM cells

The release of ICD-related DAMPs in GBM holds potential for converting tumor’s immunosuppressive microenvironment into one that can elicit a strong anti-tumor immune response. Oxaliplatin, a well-known ICD inducer in tumor cells, served as a reference to test the ICD-inducing potential of DACHPt-NPs. This was assessed by measuring extracellular ATP release, a primary marker of ICD, along with high mobility group B1 (HMGB1) release.

The effects of a range of DACHPt-NPs concentrations were evaluated in U87MG cells during the first 8 hours of treatment and ATP levels were monitored by detecting the bioluminescence generated by the enzymatic reaction of luciferase with ATP-activated luciferin. As shown in Figure 4(a), ATP levels increased markedly in the early hours of DACHPt-NPs treatment, across all tested concentrations as compared to baseline levels of untreated cells. In particular, a significant boost in ATP release was observed within the first 30 minutes up to 5 hours in cells treated with 200 μg/ml DACHPt-NPs and within the first hour in cells treated with 100 μg/ml DACHPt-NPs. Following this peak, a gradual decline in ATP levels was observed, suggesting sustained release over time. These results indicate that ATP release occurred rapidly following DACHPt-NP treatment and was dose-dependent.

Figure 4.

Release of damage-associated molecular patterns (DAMPs) related to immunogenic cell death in U87MG cells. (a) DACHPt-NPs-induced extracellular ATP release in U87MG cells. The amount of ATP released was measured in relative light units (RLU) at different time points during the first 8 hours of treatment. Differences in ATP release were observed for each concentration, with comparisons made to the ATP levels at time point 0 (n = 6). (b) Kinetics of extracellular ATP release following treatment with empty nanoparticles (blank-NPs), drug-loaded nanoparticles (DACHPt-NPs) and free oxaliplatin. RLU of cells treated with 100 μg/ml and 200 μg/ml of DACHPt-NPs, blank-NPs and free oxaliplatin were analyzed at different time points (B-left). Significant differences of DACHPt-NPs treatments compared to oxaliplatin and blank-NPs are shown with* and # symbols, respectively. All significant differences are reported in Table 1 Supplementary (n = 6). Area under the curve (AUC) analyses were carried out (B-right). (c) High mobility group box 1 (HMGB1) release by U87MG cells after 24 hours from treatment with blank-NPs, DACHPt-NPs and free oxaliplatin. In all cases, the data were normalized to the average RLU of the untreated samples, used as a negative control (n = 3). Comparisons were performed using: a two-way ANOVA followed by Dunnett’s multiple comparison test (a), a mixed-effects analysis test followed by Tukey’s multiple comparison test (B-left); an one-way ANOVA followed by Sidak’s multiple comparisons test (B-right), a two-way ANOVA followed by Tukey’s multiple comparison test (c). *p<.05, **p<.001, ***p<.01, ****p<.0001.

Subsequent testing compared the effects of free oxaliplatin to DACHPt-NPs at the same concentrations, while for blank-NPs, polymer concentrations equivalent to those in DACHPt-NPs were applied. Figure 4(b) (left) shows ATP levels across all treatments at specific time points, at the two tested concentrations. Notably, DACHPt-NPs at both concentrations significantly increased ATP release compared to empty NPs and free oxaliplatin. All these differences are reported in Table 1 supplementary. We noticed that free oxaliplatin induced minimal ATP release, similar to untreated cells. This was expected, as the maximum tested oxaliplatin dose (200 μg/ml) did not significantly reduce cell viability (IC₅₀ for oxaliplatin in U87MG cells was 290 μg/ml, Table 2). An increase in ATP release was also observed in cells treated with blank formulations, likely due to membrane permeability changes induced by nanoparticle internalization, which could cause passive ATP release. Additionally, some cytotoxic effects from high polymer concentrations (HA and PA) at 100 and 200 μg/ml may have contributed to ATP release. However, ATP release induced by DACHPt-NPs was significantly higher than that induced by blank-NPs.

To further assess extracellular ATP release over the initial 3 hours of treatment, Area Under the Curve (AUC) analyses were performed (Figure 4(b), right). Results showed that treatment with 200 μg/ml DACHPt-NPs induced significantly more ATP release, than both blank-NPs and free oxaliplatin at the same concentration.

Since HMGB1, a chromatin-binding protein, is a key marker of ICD typically released in the later stages of cell death [16], we assessed its release to evaluate the therapeutic effects of the drug-loaded nanosystem after 24 hours. Similar to the ATP release experiments, U87MG cells were treated with DACHPt-loaded NPs, blank NPs, and a free oxaliplatin solution. HMGB1 release was then measured 24 hours post-treatment.

Results showed that blank NPs induced minimal HMGB1 release across all tested doses. In contrast, free oxaliplatin consistently induced moderate HMGB1 release at each dose, while DACHPt-loaded NPs resulted in a marked increase in HMGB1 release in U87MG cells, at the highest concentration tested (100 μg/ml), as shown in Figure 4(c).

Together, these findings demonstrate the enhanced effects of the drug-loaded nanosystem on cellular internalization and the induction of ICD-related DAMPs, compared to the effects of the free drug molecule alone.

3.4. Evaluation of NPs uptake by the cells present in the GBM microenvironment and patients’ blood leukocytes

After confirming the efficient targeting of the GBM cell line and in vitro-derived macrophages with the polymeric nanosystem, we extended our evaluation to single cell suspensions derived from specimens resected from GBM patients undergoing surgery.

Previous studies, including our own, have highlighted the critical role of myeloid cells within the TME of GBM. Specifically, bone marrow-derived macrophages (BMDMs) exhibit enhanced intrinsic immunosuppressive capabilities compared to resident microglial cells (MG), and their recruitment from the periphery to the tumor core correlates with increased immunosuppressive activity [24]. During tumor progression, BMDMs are actively recruited to the tumor site, where they can be distinguished from tissue-resident MG using a combination of antibodies in multicolor flow cytometry analysis (Figure 5(a), upper panel).

Figure 5.

Uptake of cyanine 5 nanoparticles (Cy5-NPs) by cells in the glioblastoma (GBM) microenvironment and blood leukocytes from patients. (a) The gating strategy to identify cells present in the GBM microenvironment is reported. After doublets/dead cells exclusion, through the expression of CD45, immune infiltrating cells were distinguished from tumor cells. Inside the infiltrative cells (CD45⁺), subsets were gated as follows: lymphocytes as CD45⁺SSC^Low, PMNs as CD33^LowSSC^Mid, macrophages as CD33⁺ that were further characterized in BMDMs as CD49⁺HLA-DR^High and MG as CD49⁻HLA-DR^Mid. (b) Cell suspensions from GBM specimens were treated for 3 hours with 50 µg/ml Cy5-NPs or empty nanoparticles (blank-NPs) and uptake was evaluated by flow cytometry. Left- Representative histogram plot of the incorporation of fluorescent polymeric nanosystem by GBM cells; right – quantification of the Cy5 mean fluorescence intensity (MFI) in the corresponding cells subsets (n = 4). (c) Peripheral blood leukocytes were incubated with Cy5-NPs or blank-NPs for 3 hours and the uptake was evaluated by flow cytometry analysis. Cy5-NPs uptake by lymphocytes (orange), monocytes (pink), and PMNs (green) compared with cell autofluorescence (light blue) is shown in a representative sample (left) and quantification of the Cy5 MFI in the right graph (n = 4). Bars represent the mean ± SD of the Cy5 MFI. Comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Freshly resected tumor biopsies underwent mechanical and enzymatic digestion to generate a single-cell suspension, which was subsequently treated with either Cy5-NPs or Blank-NPs for a 3-hour incubation time followed by flow cytometry analysis. The analyzed cell subsets differentially incorporated the fluorescent NPs: only 30% of lymphocytes were positive for the Cy5 signal, compared to 70–90% of tumor and myeloid cells (Figure S1). In terms of mean fluorescence intensity (MFI) of Cy5, results indicate that BMDMs exhibit the highest uptake, followed by MG cells, and then tumor cells (identified as CD45⁻ subset). Polymorphonuclear cells (PMNs) exhibited very low intensity signal of Cy5, while lymphocytes demonstrated negligible uptake levels (Figure 5(b,c)).

We also investigated the uptake of polymeric nanoparticles by circulating leukocytes from the blood of GBM patients. Results showed high level of uptake by monocytes (evaluated as CD14⁺ cells) and PMNs (evaluated as CD15⁺ cells), whereas CD3⁺ cells T lymphocytes exhibited low level of Cy5 fluorescence.

The high uptake of nanoparticles by PMNs, representing the predominant circulating leukocyte subset, suggests that the systemic administration of these nanoparticles may not be a viable strategy. Nonetheless, the selective targeting of immunosuppressive myeloid cells and malignant cells within the TME indicates that a localized therapeutic approach may be more appropriate.

4. Discussion

Chemotherapy regimens using platinum-based drugs are widely applied for the treatment of several malignancies. However, their lack of selectivity, high systemic toxicity, and drug resistance severely limit their clinical application. Therefore, improvement of specific delivery of these drugs to tumors is crucial to increase therapeutic drug concentrations at pathological sites, while minimizing the side effects associated with unspecific non-targeted drug distribution [7]. In this work, we used nanoparticles loaded with DACHPt (Figure 1, Table 1) to enhance drug selectivity toward myeloid and tumor cells and simultaneously induce ICD in these last cells. The rationale for encapsulation of DACHPt is based on the need to improve its therapeutic efficacy and targeting ability toward tumor cells and tumor-promoting cells and reduce the side effects of the parent drug [21,28]. In addition, works of Cabral and others [29] have demonstrated that encapsulation of DACHPt prolonged blood circulation and increased accumulation in tumor tissue compared to free oxaliplatin.

In vitro studies using GBM cell lines and in vitro derived-macrophages as models for cells present in the GBM TME, demonstrated efficient incorporation of NPs into the cells of interest (Figure 2). Notably, we observed that the cytotoxic activity of DACHPt was enhanced through its encapsulation in polymeric NPs (Figure 3). Cytotoxic effects, as confirmed by the calculated IC₅₀ values, are exerted by the encapsulated drug and not by the empty nanoparticles (Table 2), thus suggesting a favorable safety profile. Moreover, Matha and colleagues previously conducted pharmacokinetic studies after intravenous injection (IV) of DACHPt-loaded nanoparticles in healthy mice to demonstrate that there were not any toxicity issues and that once encapsulated the drug was retained longer in the blood circulation, resulting in a higher AUC and MRT, and a lower plasmatic clearance compared to oxaliplatin [21]. To confirm ICD induction following treatment with DACHPt-NPs, we measured the release of extracellular ATP and HMGB1, two key DAMPs [30]. Both these assays provide insight into the cellular dynamics within the GBM TME and can help to assess the effectiveness of treatments targeting the tumor or modulating the immune response. As previously demonstrated in the work of Matha et al. [21], a high concentration of DACHPt-NPs significantly increased the release of HMGB1 release from pancreatic cells, but in that case no differences were observed in ATP release. In our study instead, results on GBM cell line demonstrated that encapsulated DACHPt was more effective at inducing both DAMPs release: HMGB1 and ATP, compared to the free oxaliplatin (Figure 4). This suggests that NP-based delivery can reduce the required drug concentration, potentially minimizing adverse effects in GBM.

To evaluate the clinical potential of these nanoparticles, we conducted studies with blood samples and tumor specimens from GBM patients. These studies revealed efficient internalization of the nanoparticles by key target cells such as tumor cells, BMDMs and circulating monocytes (Figure 5). However, the current study revealed significant uptake by circulating PMNs, the predominant leukocyte population in the blood. This finding indicates a limitation for systemic administration of DACHPt-NPs and suggests that a local treatment approach may be more suitable for GBM treatment. DACHPt-NPs could be administered locally in the surgical cavity after tumor resection to target and eliminate residual immunosuppressive myeloid cells and tumor cells, especially when gross total resection cannot be performed.

It is well known that GBM is resistant to immune checkpoint inhibition, in part due also to the presence of exhausted T cells, characterized by the expression of markers like programmed cell death (PD1), LAG3, TIGIT and CD39 [31,32]. The proposed nano-based approach aims to induce ICD, thereby improving the overall therapeutic efficacy. Inducing ICD through chemotherapy with immunogenic properties holds the potential to prime tumors for immunotherapy by promoting the release of DAMPs that, in turn, can activate the immune system and boost systemic anti-tumor immunity. When cancer cells undergo ICD, they release signals into the microenvironment and possibly into systemic circulation, where they reach and stimulate dendritic cells to present neoantigens to T cells – a critical step for the activation of tumor-specific T cell responses [8,9]. Nanomedicine-based chemotherapeutics can significantly improve ICD induction, by targeting tumor cells and immunosuppressive cells more precisely, thus leading to a re-activation of the anti-tumor immune response and to a more robust and sustained effect [33]. Combining nanomedicine-based chemotherapy with immunotherapy can create a synergistic effect, enhancing overall therapeutic efficacy. The work of Gu et al., indeed demonstrated the potential of enhancing anti-tumor immunity through oxaliplatin-loaded liposomes and localized immunotherapy via STING activation in mouse model of colorectal cancers [34]. In addition, other studies have shown that theranostic probes capable of crossing the Blood-Brain Barrier (BBB) and combining chemotherapy with real-time immune response tracking can enhance treatment precision and outcomes [35–37].

Advantages of using polymeric nanoparticles comprise also their ability to be customized for specific cell targeting into the brain tissue by modifying their surface with ligands such as antibodies and proteins. Temozolomide (TMZ), the first FDA-approved alkylating agent and chemotherapeutic used for GBM, is the most commonly employed small-molecule drug in GBM clinical trials since it can penetrate the BBB [38]. Indeed, M.J. Ramalho and colleagues have developed TMZ-poly (lactic-co-glycolic acid) nanoparticles that were modified with a monoclonal antibody targeting the transferrin receptor, which is known to be overexpressed in GBM tumor cells. Their study revealed a heightened inhibition of tumor cell growth when utilizing the encapsulated TMZ treatment compared to the free drug in glioma cell lines [39].

One of the polymers in the nanosystem, HA, is a glycosaminoglycan that specifically recognizes membrane glycoproteins such as CD44 receptors, which are overexpressed on various cancer and immune cells, including macrophages [40,41]. Thus, we hypothesize that the internalization of HA-decorated system may occur, at least in part, through CD44 binding, as previously demonstrated in colon, lung and pancreatic cell lines [21]. However, it is important to note that in macrophages, nanoparticle internalization is likely mediated by multiple mechanisms, such as endocytosis, pinocytosis, and receptor-mediated phagocytosis, which may work alongside CD44-mediated internalization. Therefore, while CD44 may contribute to the uptake, it is likely not the only receptor involved, especially in macrophages, which are known to utilize various internalization mechanisms.

5. Conclusions

In conclusion, this study highlights the potential of drug-loaded nanosystems to significantly enhance the delivery of cytotoxic agents to both tumor cells and tumor-associated myeloid cells, which contribute to tumor-promoting ability. The primary focus of this work was the in vitro biological validation of this nanosystem, particularly its ability to induce immunogenic cell death (ICD) in glioblastoma cells. Further investigation in preclinical models are warranted to evaluate the therapeutic efficacy and safety of DACHPt-loaded NPs in vivo, with the ultimate goal of translating this approach into clinical application for GBM patients. However, further optimization studies of nanoparticle formulation are required to achieve precise cell targeting and provide more robust therapeutic effects. Moreover, future strategies should combine nanotechnology with immune-stimulating therapies, such as immune checkpoint inhibitors. We believe that only a combined approach targeting, on one side, the immune-suppressive tumor microenvironment and, on the other side, stimulating the immune response with an immune checkpoint inhibitor (like anti-PD1), can provide a robust therapeutic effect. This combinatorial approach may effectively shift the immunosuppressive “cold” TME into a more pro-inflammatory “hot” state, thereby increasing the tumor’s responsiveness to immunotherapy [33,42,43].

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Funding Statement

This work was supported by TRANSCAN-2 ERA-NET; Università degli Studi di Padova [BIRD205873, SID 2020, DiSCOG]; and Intramural Research Funding Programs IOV-IRCCS [project RESTART] to Susanna Mandruzzato and ANR-16-IDEX-0005 Arqus, ANR project NanoImmune to Giovanna Lollo. This research received Ricerca Corrente funding from the Italian Ministry of Health to cover publication costs.

Article highlights

• Glioblastoma multiforme (GBM) is characterized by a highly immunosuppressive tumor microenvironment (TME), which impairs the effectiveness of conventional immunotherapy.

• Immunogenic cell death (ICD) triggers an immune response, enabling the immune system to recognize and attack tumor cells.

• Danger-Associated Molecular Patterns (DAMPs) released during ICD act as “eat me” signals that activate tumor-specific innate and adaptive immune responses.

• The oxaliplatin analogue DACHPt is a potent ICD inducer, presenting suitable chemical and physical properties for its encapsulation in a polymeric nanosystem.

• In this study, DACHPt-NPs are shown to induce in vitro the release of ICD-related DAMPs with significantly higher efficiency than the clinically used free drug oxaliplatin.

• Uptake studies on cells derived from dissociated GBM showed that NPs were efficiently incorporated by myeloid and tumor cells, but not by lymphocytes.

Author contributions

A Tushe: conceptualization, investigation, methodology, data curation, formal analysis, writing – original draft, writing – review and editing. E Marinelli: investigation, data curation, formal analysis, writing – review and editing. B Musca: investigation, writing – review and editing. S Zumerle: formal analysis, writing – review and editing. A Ventura, O Slukinova, G Zampardi: writing – review and editing. F Volpin, C Bonaudo, A Della Puppa: resources, M Repellin, G Guerriero: methodology, validation, writing – review and editing. G Lollo: funding acquisition, methodology, validation, writing – original draft, writing – review and editing. S Mandruzzato: conceptualization, funding acquisition, resources, writing – original draft, writing – review and editing, supervision, project administration.

All authors revised and accepted the submitted version of their work and agree to take personal responsibility for their contributions.

Disclosure statement

The authors have no 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. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Views expressed in the submitted article are from the authors and not form an official position of their institutions.

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

Ethical Declaration

Patients were recruited at the Department of Neurosurgery, Padova University Hospital and Florence University Hospitals (from 2021 to 2023). All the experiments were approved by the ethics committees of the Veneto Institute of Oncology–IRCCS of Padova, Italy (MDSC_SNC 2016/13) and the Padova and Florence University Hospitals (NOI_NCH 1536/19). All patients gave their written informed consent, and the study was conducted in accordance with the Declaration of Helsinki

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17435889.2025.2497747