Docetaxel-loaded anti-PSMA-functionalised PLGA-PEG nanoparticles suppress cell migration in LNCap prostate cancer cells and promote cell death in 3D spheroids

Introduction: Polymeric nanoparticles have been used in drug delivery to treat cancer by selective targeting of tumour cells and inducing programmed cell death. Previously optimized nano-systems loaded with docetaxel and functionalized with anti-PSMA antibody were used to investigate targeting of prostate cancer cells using prostate-specific membrane antigen (PSMA) as a target for receptor-mediated endocytosis and enhanced cellular uptake.

Methods: Cell migration, apoptosis, cell cycle, and generation of reactive oxygen species were evaluated using in vitro assays on PSMA-positive LNCap and PSMA-negative PC-3 prostate cancer cells. Thereafter, the growth inhibition and viability of 3D spheroids cultured from LNCap cells were measured, and the efficacy of the targeted nano-system and its interaction with the spheroids were investigated using fluorescence microscopy.

Results: In LNCap cells, the targeted system outperformed the non-targeted counterpart, resulting in migration inhibition of 89% ± 8.4% versus 48.1% ± 2.7% and G2/M phase arrest of 30.3% ± 3.0% versus 15.8% ± 2.8%. Conversely, treatment of PSMA-negative PC-3 cells resulted in no statistically significant changes to migration or cell cycle progression (p > 0.05), indicating that the therapeutic potency of the targeted system is mediated by PSMA-receptor-mediated endocytosis, a pathway unavailable in PC-3 cells. There was also higher inhibition of 3D LNCap spheroids by both nano-systems than by free docetaxel, with 67.8% ± 5.5% and 17.6% ± 4.6% inhibition for the targeted and non-targeted systems, respectively. Fluorescence microscopy showed that the targeted system was able to penetrate the 3D spheroid structure and cause cell death.

Discussion: The enhanced cellular activity and spheroid penetration of the targeted nano-system in LNCap cells, together with the lack of effect in PSMA-negative PC-3 cells, support PSMA-receptor-mediated endocytosis as the key uptake mechanism and suggest the potential of this anti-PSMA-functionalised PLGA-PEG nano-system as a targeted nanocarrier for docetaxel in prostate cancer.

1 Introduction

Nanomaterials are widely adopted in the design of drug delivery systems to improve the bioavailability of drugs (Hsu et al., 2023), with cancer nanomedicine being the most studied due to its complex pathology (Mundekkad and Cho, 2022). Prostate cancer is the most common diagnosis of male carcinoma worldwide (Sung et al., 2021), and docetaxel is the anti-cancer drug of choice approved by the US FDA (Rivero-Buceta et al., 2019; Lima et al., 2021) for prostate cancer (Li et al., 2018), but it has numerous side-effects (Autio et al., 2018; El Rassy et al., 2018) and poor bioavailability due to its physicochemical properties (Li et al., 2018).

Therefore, this research proposes the evaluation of a targeted anti-body-functionalised PLGA nanosystem to bind to a prostate cancer cell surface receptor for receptor mediated endocytosis (Muhamad et al., 2018). The characteristics of prostate-specific membrane antigen (PSMA), a type II transmembrane protein overexpressed on the majority of prostate cancer cells (Wüstemann et al., 2019), were harnessed.

The targeted nanosystem, as described and evaluated in our previous work (Essa et al., 2025), showed enhanced in-vitro efficacy in 2D cell culture. The following work explores the comparative efficacy of the nano-systems on cancer associated intra-cellular processes such as cell cycle arrest, apoptosis induction and the activation of reactive oxygen species (ROS) in 2D cell culture. However, it has been reported that the interaction of nanoparticles with cellular compartments in the tumour microenvironment can hamper their transport to tumour cells (Chan, 2023), which can cause reduced efficacy in vivo. Therefore, a 3D multicellular tumour cell spheroid model was developed to better correlate with in-vivo data (Friedrich et al., 2009) by closely mimicking the heterogenous environment of in-vivo prostate cancer tumours (Bromma et al., 2022).

While PSMA-targeted delivery systems have been explored using various approaches including small molecule ligands, folic acid conjugates (Karandish et al., 2016), and antibody functionalization (Rivero-Buceta et al., 2019), and PLGA nanoparticles have been developed for docetaxel delivery, no prior study has systematically evaluated anti-PSMA antibody-functionalized PLGA-PEG-docetaxel nanoparticles with direct PSMA + versus PSMA− comparative assessments. Our specific antibody-PLGA architecture, combined with integrated mechanistic and tumor microenvironment evaluation, addresses critical translational gaps in prostate cancer nanotherapy. Here we also employ light field microscopy—a scanning-free volumetric imaging approach—to visualize real-time distribution of fluorescent nanoparticles within the spheroid architecture. This rapid 3D imaging modality, which captures complete four-dimensional light field information through a microlens array, provides complementary spatiotemporal resolution for monitoring dynamic nanoparticle-tumor interactions (Massaro et al., 2022).

Our work provides a unique evaluation using a specific anti-PSMA PLGA-PEG nanoparticle design. The direct comparison between PSMA-positive (LNCap) and PSMA-negative (PC-3) effects, and the integration of match 2D and 3D experimental readouts offers a detailed perspective on how this specific nanoparticle architecture overcomes biological and physical barriers of the tumor microenvironment. The value of our systematic integration approach is highlighted by recent analyses in pharmaceutical sciences demonstrating that incremental innovation through rigorous comparative validation often provides greater clinical impact than isolated platform novelty (Yin, 2023). By comprehensively characterizing our specific nanoparticle design across complementary biological models, we provide actionable data that studies focused on single platforms or mechanisms cannot deliver.

2 Materials and methods

2.1 Materials

The polymeric materials Poly (D,L-lactide-co-glycolide) (PLGA) (lactide/glycolide ratio of 50:50), molecular weight 7 000–17 000 with acid end groups, carboxylic acid polyethylene glycol PLGA (carboxylPEGPLGA), Pluronic F127, 2-(N-morpholino)ethanesulfonic (MES) free acid, Tris (hydroxymethyl)aminomethane (TRIS),1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide free acid (NHS), Anti-PSMA antibody, hydroxylamine hydrochloride, sodium hydroxide pellets (NaOH), hydrochloric acid, fluorescein iso-thiocyanate (FITC), Hoescht-3342, 4′,6-Diamidino-2-Phenylindole (DAPI), tween80, and acetonitrile, were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Pty) Ltd, Estate South, Modderfontein, Gauteng, SA. Docetaxel was obtained from DLD Scientific, Durban South Africa. For cell culture and experiments, Roswell Park Memorial Institute (RPMI) and Dulbeccos’s Minimum Essential (DMEM) culture medium, fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Thermofisher Scientific, Waltham, MA, USA.

2.2 Preparation and characterization of nanoparticles

The preparation and characterisation of antibody-conjugated nanoparticles was described previously (Essa et al., 2025). Briefly, PLGA, or a mixture of PLGA and carboxylPEGPLGA, and docetaxel dissolved in acetonitrile served as the organic phase, while a 0.1% solution of Pluronic F127 in Millipore water served as the aqueous phase for fabrication of nanoparticles using the NanoAssemblr Benchtop (Precision Nanosystems Inc., Vancouver, BC, Canada. The resultant suspensions were stirred for 4 h at room temperature and pellets were obtained by three times centrifugation at 12,000 rpm for 30 min. PLGA-docetaxel nanoparticles, were the non-targeting nanosystem (NTN) while active targeting nanoparticles (ATN) were further functionalized by conjugation of the PLGA: PLGA-PEG-COOH with anti-PSMA antibody via carbodiimide chemistry. Fluorescent nanoparticles, called FITC-ATN were prepared in the same way, except FITC was used instead of docetaxel in the organic phase, and the preparation was conducted in the dark. The nanoparticle pellets were frozen at −80 °C and then freeze dried (Freezone 12 lyophilizer, Labcono, Kansas City, USA) for 24 h.

2.3 Cell culture conditions

PC-3 and LNCap prostate carcinoma cells were obtained from Cellonex (Johannesburg, South Africa). Cells were confirmed by supplier to be free of mycoplasma. PC-3 cells were grown in RPMI culture medium, and LNCap cells were grown in DMEM/F12 culture medium (Gibco). The media were supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) as per cell culture protocols. The cells were cultured under sterile conditions in an incubator at 95% humidity and 5% CO2. For all assays, cells were counted using the LUNA II Automated Cell Counter (Logos Biosystems, Anyang-Si, South Korea) before seeding. A concentration of 10% DMSO was used as a positive control in cytotoxicity, apoptosis, cell cycle arrest, and spheroid growth inhibition assays. Cell culture medium was used as a negative control in these experiments.

2.4 Cancer cell scratch assay

PC-3 and LNCap cells were grown in RPMI and DMEM/F12 media, as prepared above and grown until 80% confluent. Thereafter, they were harvested and seeded in six well plates at a density of 2.5 × 104 cells/well for PC-3 and 5 × 104 cells/well for LNCap and grown until 80% confluence. Thereafter a scratch was made across each well with a 100 µL pipette tip, and cells were treated with whole media (control), and docetaxel, NTN and ATN. The minimum effective concentration of docetaxel (8 ng/mL) was determined previously (11) and confirmed during the assay timeline. Docetaxel was dissolved in DMSO and diluted to a final treatment concentration of 0.1% DMSO, which was also used as the vehicle control. Images were captured every 24 h over a 72-h period, and gap closure inhibition relative to untreated control was evaluated using ImageJ software, using Equations 1,2. adapted from (14). The workflow of the assay is shown in Figure 1, adapted from (Grada et al., 2017).

$\%woundclosure=\frac{\left(W{t}_0-W{t}_x\right)}{W{t}_0}\times 100\%(1)$

Figure 1

Figure 1. Workflow of the scratch assay conducted on PC-3 and LNCap (adapted with permission from Grada et al.).

Where Wt₀ is the initial wound area at time t0 and Wt_x is the wound area at time x, the time point of interest.

$\%closureinhibition=\left(\%WC{c}_x-\%WC{t}_x\right)(2)$

Where WCc_x is the wound closure of the control group and WCt_x is the wound closure of the treated group at time x.

2.5 Analysis of apoptotic effect and cell cycle using the MUSE™ Cell Analyzer

The MUSE™ Cell Analyzer (LuminexCorp, Austin, TX, USA) was used to detect miniaturized fluorescence emission from single cell suspensions in order to provide quantitative information on the effects of the nanoparticle treatments on apoptosis and the cell cycle of PC-3 and LNCap cells. A system check and complete clean cycle were run prior to experimentation. Additionally, the quick clean function was carried out in between experiments to prevent sample cross contamination and blockage of the capillary tubes.

2.5.1 Apoptotic effect by annexin V and propidium iodide reaction

The apoptotic effect was assayed using the MUSE™ Annexin V and Dead Cell Kit (LuminexCorp, Austin, TX, USA). PC-3 and LNCap cells were seeded at 1 × 104 cells/mL and treated with concentrations of docetaxel equivalent to effective concentration of 8 ng/mL of native docetaxel, NTN’s and ATN’s. After 72 h, cells were washed with PBS, trypsinized for 4 min, and diluted with culture media. Thereafter, 100 µL samples of cell suspensions were aliquoted into microcentrifuge tubes and 100 µL of the MUSE™ Annexin V and Dead Cell Reagent was added to each tube, which was then vortexed gently for 5–10 s to homogenize. Samples were stained for 20 min at room temperature in the dark and then analyzed by the MUSE™ Cell Analyzer by selecting the Annexin V and Dead Cell Assay.

2.5.2 Cell cycle analysis

The cell cycle was analysed using the MUSE™ Cell Cycle Kit (LuminexCorp, Austin, TX, USA). PC-3 and LNCap cells were seeded at 1 × 104 cells/mL and treated. After 72 h, cell suspensions were centrifuged, washed with PBS and fixed with cold 70% ethanol. Samples were frozen at −20 °C for at least 4 h. Thereafter, samples were centrifuged at 300 g for 5 min, washed once with PBS and thereafter, the resulting cell pellet was aliquoted into microcentrifuge tubes. 200 μL of the MUSE™ Cell Cycle Reagent was added to each tube, which was then vortexed. Samples were stained for 30 min in the dark and then analyzed by the MUSE™ Cell Analyzer by selecting the Cell Cycle Assay.

2.6 Determination of ROS activation

Reactive oxygen species in cells were determined using the Fluorometric Intracellular ROS kit (Sigma-Aldrich, St. Louis, MO, USA). LNCap and PC-3 cells were seeded in 96 well plates at a cell density of 5 × 103 cells/well in DMEM/F12 and RPMI respectively. Twenty-four hours post cell attachment, cells were treated with 100 µL of the fluorometric master buffer assay mix and incubated for 1 h. Thereafter cells were treated with growth media (control) and the effective concentration of 8 ng/mL of docetaxel, NTN and ATN. Plates were read 72 h after treatment at excitation wavelength of 475 nm and emission wavelength of 520 nm. Fluorescence intensity was taken to be proportional to ROS activation as per assay protocol.

2.7 Spheroid optimization assay

Spheroids were generated from the LNCap cell line using a method adapted from (Friedrich et al., 2009). Cell culture plates were coated with 1% agarose in PBS, and cell were seeded over this at different densities, with five replicates per density. Spheroids were monitored using bright field microscopy, and volumes were calculated using Equation 3 (16).

$V=a\left(\frac{b^2}{2}\right)(3)$

Where V = volume of spheroid in µm³, a = major diameter in µm and b = minor diameter in µm.

2.8 Spheroid viability assay

Spheroids were measured on day 4 and treated with a range of concentrations of docetaxel, NTN and ATN. A treatment of 5% DMSO was used as a positive control for cell death. Seventy-two hours later (day 7), Alamar Blue assay reagent was added to the culture medium, and culture plates were incubated for 3 h at 37 °C. Thereafter, fluorescence readings were taken using 535 nm excitation and 595 nm wavelengths on the Viktor X3 multiplate reader (Perkin Elmer, MA, USA).

2.9 Spheroid growth inhibition assay

Spheroids were imaged and measured on day 4 and treated with media (control) docetaxel, NTN and ATN. Spheroids were measured 72 h later (day 7) and treated again. Thereafter, spheroids were treated and measured every 48 h (days 9, 11, 13, 15), and measured again at day 17. which was the endpoint of the assay. This schedule is adapted from the work by Friedrich and co-workers (Friedrich et al., 2009).

2.10 Live/dead spheroid fluorescent viability assay

Experiments were conducted in the dark. Spheroids were seeded and treated with media (control) docetaxel, NTN and ATN. After 72 h, spheroids were washed three times with PBS and stained with 2 µm calcein and 4 µm ethidium homodimer using the LIVE/DEAD viability/cytotoxicity kit (Thermofisher, Waltham, MA, USA), according to the method by Petry and Salzig (17). The spheroids were counterstained using a 1:10,000 dilution of 1 mg/mL Hoescht 33,342 and incubated for 30 min at 37 °C. Thereafter, spheroids were imaged with the CELENA X Digital Imaging System (Logos Biosystems, Anyang-Si, South Korea) using the DAPI, EGFP and RFP fluorescent channels. Images were then processed using ImageJ sotware.

2.11 Confocal microscopy of nanoparticle treated spheroids

Experiments were conducted in the dark. Spheroids were seeded and treated with FITC-ATN for 24 h. They were then collected in microcentrifuge tubes, washed thrice with PBS, and fixed using 4% paraformaldehyde for 30 min. Following three further PBS washes, nuclei were stained with 1 μg/mL DAPI for 10 min. The spheroids were then mounted onto glass slides for viewing on the Zeiss LSM910 Lightfield 4D System with AiryScan 2 (Zeiss, Jena, Germany).

2.12 Statistical analysis

All experimental data were processed and analyzed by Origin software (version 8.5.0 SR1. OriginLab Corporation, U.S.). All results were represented by the mean ± standard error of three biological and three technical replicates, unless otherwise stated. Comparison of data between experimental groups was evaluated by one way analysis of variance (ANOVA) and Bonferroni adjustment, with p values less than 0.05 being designated as significant.

3 Results

3.1 Assessment of nanosystem therapeutic efficacy by cancer cell scratch assay

Here, images were captured at 0H (time of scratch) to measure the initial wound area, and thereafter the change in wound area was measured and calculated by Equations 1 and 2. Every 24 h for each treatment on each cell line. For the assay on PC-3 cells, all treatments had no significant difference in wound closure at 24 h and cells migrated to fill the wound area by 48 h (Figure 2). This indicated that none of the treatments had any noticeable or significant effect on wound closure.

Figure 2

Figure 2. Wound healing of PC-3 cells treated with growth media (control), and effective concentrations of 8 ng/mL docetaxel in free docetaxel (DOC), non-targeted nanoparticles (NTN) and targeted nanoparticles (ATN).

As can be seen by Figures 3, 4A,C, the migration rate of both treated and control groups of LNCap cells was much slower than PC-3 cells, with the control group only reaching 61.0% ± 7.4% wound closure after 72 h. This is consistent with a recent study by Bossio and co-workers (Bossio et al., 2023) who achieved <75% wound closure of the control group of LNCap cells after 72 h. In this study, the NTN treated group showed significantly greater inhibition of closure than the docetaxel treated group at 48 (39.0% ± 1.1% for NTN and 24.6% ± 0.7% for docetaxel) and 72 h (48.1% ± 2.7% for NTN and 11.6% ± 7.1% for docetaxel), but displayed no significantly different inhibition percentage to docetaxel at 24 h, shown by Figure 4B. This could be due to the initial availability of the entire dose of docetaxel in the solution media, compared to the NTN system which showed a sustained release profile (11) and therefore caused more inhibition at the later time points.

Figure 3

Figure 3. Wound healing of LNCap cells treated with growth media (control), and effective concentrations of 8 ng/mL docetaxel in free docetaxel (DOC), non-targeted nanoparticles (NTN) and targeted nanoparticles (ATN).

Figure 4

Figure 4. Percentage Wound Closure and Wound Closure Inhibition relative to control of PC-3 (A,B) and LNCap cells (C,D) after being treated for 24. 48 and 72 h with growth media (control), and 8 ng/mL of docetaxel in free docetaxel (DOC), non-targeted nanoparticles (NTN) and targeted nanoparticles (ATN). (*p value < 0.05).

3.2 Apoptotic effect of treatments

The apoptosis profiles of treated groups are reflected in Supplementary Figure S1 for PC-3 cells and Supplementary Figure S2 for LNCap cells. The main apoptotic pathways measured were through early and late apoptosis, and there was no significant difference in the total apoptosis of native docetaxel (4.83% ± 0.4%), NTN (3.4% ± 0.80%) and ATN (4.25% ± 0.63%) in PC-3 cells. However, both nanosystems displayed lower necrosis profiles (top left quadrant in Supplementary Figure S1), with 2.27% ± 0.37% for NTN and 1.26% ± 0.07% for ATN than free docetaxel (4.12% ± 0.30%). There was no significant difference between the apoptotic profiles of both nanosystems on PC-3 cells.

Interestingly, there was also no significant difference in the apoptotic profiles and total apoptosis of both nanosystems on LNCap cells (8.32% ± 0.68% for NTN and 9.01 ± 0.90 for ATN), as shown by Figure 5, but both were significantly greater than the total apoptosis of free docetaxel, which was 3.82% ± 0.38%.

Figure 5

Figure 5. Profiles showing apoptotic populations of LNCap and PC-3 cells after being treated for 72 h with growth media (control), and 8 ng/mL of docetaxel in free docetaxel (DOC), non-targeted nanoparticles (NTN) and targeted nanoparticles (ATN). (*p value < 0.05).

3.3 Cell cycle arrest

As suggested by Supplementary Figure S3 and reflected in Figure 6, there was no significant difference in the percentages of PC-3 cells in the G2/M phase across all treatment groups, indicating that none of the treatments could cause the G2/M phase mitotic arrest that is usually associated with docetaxel.

Figure 6

Figure 6. Comparison of populations in the different cell cycle phases of LNCap and PC-3 cells after being treated for 72 h with growth media (control), and 8 ng/mL of docetaxel in free docetaxel (DOC), non-targeted nanoparticles (NTN) and targeted nanoparticles (ATN). (*p value < 0.05, one-way ANOVA).

There was a significantly higher percentage of LNCap cell populations in the G2/M phase of both the groups treated with the nanosystems, compared to the untreated control and native docetaxel treated groups, as shown in Figure 6. The G2/M phase populations of the control and docetaxel treated groups which were not significantly different to each other, which implies that at this concentration, free docetaxel was unable to cause significant mitotic arrest. However, there were significantly higher percentages of G2/M phase populations in the NTN treated (36.4% ± 1.0%) and ATN treated (50.9% ± 1.2%) groups, as shown in Figure 6.

3.4 ROS activation

The ROS activation was analysed fluorometrically and the results showed no significant difference in the ROS activation levels between any of the treated groups relative to the control in PC-3 cells, as shown in Figure 7. However, the ROS activation levels of both nanosystems were significantly greater than the control and docetaxel in LNCap cells, while the level of the NTN treated group was not significantly different to the ATN treated group, also reflected in Figure 7.

Figure 7

Figure 7. Activation of reactive oxygen species of PC-3 and LNCap cells after being treated for 72 h with growth media (control), and 8 ng/mL of docetaxel in free docetaxel (DOC), non-targeted nanoparticles (NTN) and targeted nanoparticles (ATN). (*p value < 0.05).

3.5 Generation of LNCap multi-cellular tumour spheroids

In this study the seeding density of LNCap cells was optimized to generate uniform spheroids, using 10,000 to 650 cells per well (Supplementary Figure S4). Singular spheroids did not grow with the seeding density of 10,000 cells per well, while the spheroids that developed from the initial seeding density of 5000 cells/well displayed irregular morphology, as shown in Supplementary Figure S4. The formation of a necrotic core can be observed in some of the spheroids initially seeded with 5000 cells/well.

3.6 Spheroid viability assay shows reduced response to treatments

As Figure 8 shows, free docetaxel was not effective at reducing the viability of the spheroids at any of the tested concentrations, while the NTN treatment was significantly more effective than docetaxel alone at concentrations of 40 ng/mL and 50 ng/mL. However, the ATN treatment was significantly more effective than both treatments at 20 ng/mL and 30 ng/mL. For this study, 20 ng/mL was used as the effective concentration of free docetaxel and docetaxel in nano-systems for all further spheroid assays.

Figure 8

Figure 8. Viability of LNCap Multicellular Tumour Spheroids after being treated for 72 h with a range of concentrations of docetaxel, NTN’s and ATN’s (*p < 0.05, using One-way ANOVA).

3.7 Spheroid growth inhibition assay

Treatments were conducted on days 4, 7, 9, 11 and 13. As shown by Figure 9, there was no observable difference between the morphology of untreated spheroids and the spheroids treated with docetaxel at all time points. Spheroids treated with the NTN showed cell death from day 11, while those treated with the ATN showed cell death from day 7.

Figure 9

Figure 9. Bright field images showing 17-day growth of LNCap multicellular tumour cell spheroids treated with growth media (control), and 20 ng/mL of docetaxel in free docetaxel (DOC), non-targeted nanoparticles (NTN) and targeted nanoparticles (ATN). Five replicates were conducted for each treatment condition.

Figure 10 shows that both untreated and spheroids grew exponentially, at a much faster rate than both the nano-system treated groups after day 9. This has been observed in previous inhibitory studies on spheroid growth (Brüningk et al., 2020; Kulesza et al., 2023). There was also no significant difference on spheroid growth between the untreated spheroids and docetaxel treatment at all time points. The endpoint of the assay was recommended to be at any point after 14 days, when the volume of the untreated spheroid reaches five times the volume before first treatment (Friedrich et al., 2009). In this study, this volume translated to 0.414 mm3 on day 15. Thereafter, one more measurement was taken at 17 days of spheroid growth.

Figure 10

Figure 10. Spheroid volume increase and percentage growth inhibition relative to control of spheroids treated with growth media (control), and 20 ng/mL of docetaxel in free docetaxel (DOC), non-targeted nanoparticles (NTN) and targeted nanoparticles (ATN). DMSO (5%) was used as a positive control for cell death. Five replicates were conducted per treatment condition. Values reflected are the mean of five measurements with error bars showing standard error of the mean. (*p value < 0.05, using one-way ANOVA).

As can be seen by Figure 10, there was significant inhibition of spheroid growth in the ATN treated group from day 11 (32.7% ± 4.5%) until day 17 (67.8% ± 5.5%) and in the NTN treated group from day 13 (21.0% ± 3.9%) until 17 (17.6% ± 4.6%).

3.8 Live dead fluorescence assay shows cell death caused by actively targeted nanoparticles

Figure 11 shows the staining of live cells in the media, docetaxel, and NTN treated groups This is due to calcein, which is emitted by live cell esterase. The ATN treated group shows majority of the spheroid stained with ethidium homodimer, which corresponds to the disrupted cell membranes of dead cells. The nuclei are counterstained with Hoescht 33,342 in blue, and show a reduction in the size of the spheroids in the ATN group compared to the other treatment and control groups.

Figure 11

Figure 11. Live dead spheroid assay showing viability reduction due to treatment with ATN system. CONT = untreated control, DOC = 20 ng/mL free docetaxel, NTN = non-targeted nanoparticle treatment and ATN = active targeted nanoparticle treatment. Blue fluorescence is due to intact nuclei, green fluorescence is due to live cells, and red fluorescence is due to dead cells in the spheroid structure.

3.9 Confocal microscopy shows distribution of fluorescent ATN system throughout the 3D spheroid structure

To determine if the efficacy of the ATN nanoparticles could be due to its penetration into the spheroids, we treated the group with fluorescent FITC-bound ATN nanoparticles. As Figure 12 shows, there is a distribution of the nanoparticles, shown in green, throughout the 3D structure.

Figure 12

Figure 12. Stained spheroids showing ATN nanoparticles in spheroid structure, where (A) has spheroid cell nuclei stained with DAPI, shown in blue, and (B) shows FITC-bound ATN nanoparticles in green.

Figure 13 shows the spheroid at different angles on the Y-axis, demonstrating that the nanoparticles are found throughout the spheroid surface and internal structure. This can also be viewed in the Supplementary Video S2, which shows the 3D rendering of the rotation of the spheroid along the Y-axis.

Figure 13

Figure 13. 3D images of whole spheroids with varying angles along the Y-axis. Spheroids are counterstained with DAPI in blue and FITC-ATN nanoparticles in green.

4 Discussion

4.1 Cell migration

Cell migration is involved in many important cellular processes, including cell division, tumour cell invasion, angiogenesis and metastasis. The cytoskeleton of microtubules, which are tube-like intracellular structures that promote cell division, is implicated in cell migration. This cytoskeleton should be initially intact, and thereafter, the asymmetry of microtubules precedes directional migration (Wang et al., 2019).Taxanes such as docetaxel are potent microtubule stabilizers and have been found to influence migration by interacting with microtubules and interfering with their dynamics within the cell (Bahar and Yoon, 2021).

Cancer cell scratch wound healing assays can provide insight into the therapeutic efficacy of cancer treatments as the deregulation of cell migration can affect the above physiological processes associated with cancer cell phenotypes. The tracking of the inhibition of wound closure of cancer cells after being exposed to effective concentrations can indicate the metastatic potential of the treatment (Bahar and Yoon, 2021). The assay is used to evaluate cancer cell metastasis by the inhibition of “wound healing” caused by the treatments and the time required for healing (Bouchalova and Bouchal, 2022).

Wang and co-workers obtained similar closure results after 24 h of treatment with 1 nM (0.81 ng/mL) docetaxel (Wang et al., 2022). However, they also reported marked inhibition of wound closure on PC-3 cells, 24 h after being treated with 0.1 µM (80.8 ng/mL) of docetaxel (He et al., 2024). This signifies that docetaxel could have anti-migratory effects on PC-3 cells at higher concentrations, especially since the effective concentration in this study (8 ng/mL) was to the order of 10 lower than that used in He’s study and there was no effect of any of the treatments on migration, which could be due to the relatively low effective concentration of docetaxel used.

As shown by Figure 4D, the inhibition of wound closure by the ATN treated LNCap cells was significantly greater (p < 0.05, one-way ANOVA) than the other treated groups at all time points, with 49.8% ± 3.9%, 76.7% ± 6.8% and 89.4% ± 8.4% inhibition at 24. 48 and 72 h respectively. This can be attributed to the active targeting antibody moiety on the surface of the nanosystem that could have allowed for increased cellular uptake, facilitated by the PSMA receptors found on the surface of LNCap cells, leading (Wüstemann et al., 2019) to the intracellular inhibition of motility.

The earlier observation of no effect on wound closure of any of the treatments on PC-3 cells in Figures 2, 5A,B was attributed mainly to the PC-3 cell line being insensitive to the concentration of docetaxel used. The specific biological differences between the LNCap and PC-3 cell lines, such as androgen dependence and metastatic potential, are known to influence baseline migratory potential in cancer cell lines. For example, the work by Bose demonstrated that the upregulation of androgen receptors can modulate cell migration in cervical cancer (Bose et al., 2025), while Mehanna’s group recently described how metastatic potential dictates the rate of wound closure in breast cancer models, comparing MCF-7 and MDA-MB-231, which are cell lines with low and high metastatic potential respectively (Mehanna et al., 2025). In the context of prostate cancer, the PSMA-positive profile of LNCap and the PSMA-negative status of PC-3 are well-documented characteristics, confirmed in previous studies by Western blot (Ghosh et al., 2005), fluorescence-activated cell sorting (Feldmann et al., 2017), and immunohistochemistry (Runge et al., 2023). While the observed differences in migration and cell cycle arrest between the 2 cell lines in our study do reflect the intrinsic properties of the cell lines, the efficacy of the targeted versus non-targeted system within the LNCap line warrants further consideration. The targeted system achieved a significant 41% increase in migration inhibition over the non-targeted system in LNCap cells, whereas none was observed in the PC-3 model. It is highly probable that the presence of PSMA receptors on LNCap cells facilitates a more efficient cellular uptake of the system, potentially resulting in a higher intracellular release of docetaxel. However, given the biological complexity of these cell lines, the observed enhanced efficacy is likely a synergistic outcome of both the targeted delivery mechanism and the intrinsic sensitivity of LNCaP cells.

As shown by Figure 3, from 24 h after treatment, the wound spaces of the control and docetaxel treated LNCap cells are mainly occupied by cell projections from the edge of the wound, consistent with chemotaxis, which is an important mechanism in cell migration caused by functional chemical signals (SenGupta et al., 2021). However, from the 48-h time point, there is considerable cell shrinkage and detachment from the culture plate in the wound space of the NTN and more prominently in the ATN treated groups. This phenomenon is consistent with previous cancer cell migratory inhibition studies (Wang et al., 2019; Mishra et al., 2020), and is due to enhanced cellular uptake, toxicity and inhibition of intracellular physiological processes by the nanosystems.

Taxanes have been found to inhibit wound closure at concentrations lower than effective anti-proliferative concentrations (Wang et al., 2019; Lu et al., 2020), and this is consistent with the results shown in Figure 4, which still shows low inhibition of wound closure of free docetaxel on LNCap cells even though the effective concentration of native docetaxel used is much lower than reported anti-proliterative concentrations (Edmondson et al., 2016).

4.2 Analysis of apoptotic effect of the nanosystems

Cell death can proceed through different mechanisms, the most well-known being necrosis and apoptosis (Carneiro and El-Deiry, 2020; Liu and Jiao, 2020; Chaudhry et al., 2022). Therefore, the evaluation of apoptotic potential of treatments on cancer cells can provide information about their therapeutic efficacy (Lee and Lee, 2021). Yang and co-workers (Yang et al., 2019) reported low response levels when treating PC-3 cells with low concentrations of docetaxel and only achieved higher apoptotic levels (>10%) after treating with their measured inhibitory concentration of docetaxel. The results here, at the effective concentration of 8 ng/mL docetaxel correlates with the result of no observable inhibition of wound healing obtained with the scratch assay in Figures 2, 4A,B, suggesting that apoptosis could be a driving force in the inhibition of cell migration and motility in PC-3 cells.

The fact that there was greater apoptosis by the nanosystems compared to docetaxel in LNCap cells but not in PC-3 cells could be due to the greater sensitivity of the LNCap cell line to lower docetaxel concentrations, as described previously (Kim et al., 2021). The increase in total apoptosis of the nanosystems relative to docetaxel could be the effect of improved metabolic stability of the drug within the media and sustained intracellular release (Essa et al., 2025), ensuring a prolonged therapeutic concentration of docetaxel directly at the target site within the cells. This is distinct from any targeting ability, evidenced by the lack of difference in the total apoptosis between the targeted and non-targeted systems. These results are relatively consistent with several studies which show enhanced apoptotic mechanisms of nanosystems compared to free docetaxel (Chen et al., 2016; Rivero-Buceta et al., 2019; Bromma et al., 2022). In this study, the increase in apoptosis caused by both nanosystems on LNCap cells correlates with the change in cell morphology and detachment of the monolayer observed in the scratch assay and noted in Figure 3 on the NTN and ATN treated groups. This indicates that apoptosis could be a contributing factor to this phenomenon, but since the result is more pronounced in the ATN treated group, there could be factors other than apoptosis, driven by effects such as cell cycle arrest and inhibition of proteins linked to metastasis (Vargas-Accarino et al., 2021) at work here.

4.3 Cell cycle arrest

The anti-cancer mechanism of docetaxel is well characterised (Imran et al., 2020; Ehsan et al., 2022; Hashemi et al., 2023). In this study, the cell cycle profiles of both cell lines were evaluated with all treatments to compare the mechanism of action of docetaxel and the nanosystems, and their corresponding effects on the cell cycle. Lv and co-workers reported a modest increase from 20% to 30% in the G2/M population of PC-3 cells treated with 20 nM (16.1 ng/mL) docetaxel (Lv et al., 2017), but it was not stated if this difference was significant, while Cancino’s group (Cancino-Marentes et al., 2021) and Meidavilla-Varela’s group (Mediavilla-Varela et al., 2009) both reported no significant differences when treating PC-3 cells with 25 nM (20.2 ng/mL) and 3 µM (2 424 ng/mL) docetaxel respectively. In this work, the fact that mitotic arrest did not occur correlates with the results obtained for the wound healing assay where there was no inhibition of cell migration and wound closure across all treatments in the PC-3 cell line.

Lima’s group reported a 36% increase (Lima et al., 2021) and Lv’s group reported a 47% increase (Lv et al., 2017) in the LNCap G2/M phase populations, both groups having treated with 20 nM (16.1 ng/mL) docetaxel. This could suggest that for the LNCap cells in this study, the concentration of free docetaxel used (8 ng/mL) was too low to induce cell cycle arrest in the G2/M phase. Similar results were obtained by Cao and co-workers, who prepared non-targeted and targeted PLGA nanoparticles loaded with docetaxel, and reported 30% and 60% of G2/M phase populations of LNCap cells treated with the targeted and non-targeted nanosystems respectively (Cao et al., 2016). In this study, the increase in G2/M phase populations indicate the proportional induction of mitotic arrest in the NTN and ATN treated groups. The populations translated to 15.8% ± 2.8% and 30.3% ± 3.0% arrest relative to the control for NTN and ATN treated cells, respectively. The difference in efficacy can be attributed in part to the targeting ability of the ATN, allowing for increased cellular uptake via the interaction between the targeting antibody and the PSMA receptors on the surface of the LNCap cell line, whereas the NTN does not have anti-PSMA ligands on its surface and therefore is not able to take advantage of the PSMA receptors on the LNCap cell surface. This could explain the proportional increase (ATN > NTN > DOC) in the inhibition of cell migration observed earlier, as mitotic arrest caused by prevention of microtubule polymerisation leads to inhibition of cell motility (Nakonechnaya and Shewchuk, 2015). A contributing factor is that the cell cycle arrest would have disrupted the G2/M checkpoint that prevents cells with damaged DNA from undergoing mitosis (Ventura and Giordano, 2019). Additionally, the G2 checkpoint arrest could have inhibited the formation of maturation/mitosis promotion factor (MPF), which promotes cells from the G2 to the M phase in the cell cycle (Zhang et al., 2021), thereby preventing cell migration.

4.4 ROS activation by the nanosystems

ROS is a normal by-product of healthy physiological processes (Katerji et al., 2019). However, overproduction of ROS can trigger apoptosis and therefore the measurement of the production of ROS induced (ROS activation) caused by proposed cancer treatments can be a valuable indicator of their apoptotic potential (Zaidieh et al., 2019). In this study, we observed superior ROS activation of both nanosystems compared to docetaxel but there was no observable increase provided by the targeted versus the non-targeted system. The results of the ROS activation by both nanosystems correlate with the apoptosis results which show no significant difference between the nanosystems but more apoptosis of both nanosystems compared to docetaxel.

4.5 Spheroid initiation and viability

3D multicellular spheroids have been shown to mimic the characteristics of in vivo tumours more accurately than 2D models and there has been widespread discussion of the clinical relevance of the use of in vitro 3D models in studying tumour biology (Filipiak-Duliban et al., 2022). Additionally, the 3D structure of cancer cell spheroids in assays to test efficacy of treatments are more indicative of their projected performance in vivo, as the treatment is only able to penetrate outer layers of the spheroids, much like in in vivo tumours and unlike 2D cell culture where the treatments are available in the growth medium, to a higher percentage of the cells attached to the culture plate (Perche and Torchilin, 2012). The necrotic zone indicates the inner areas of the spheroid which do not have access to oxygen and nutrients as the outer layers do, and therefore form a zone of toxic waste products (Dini et al., 2016).

The selection of effective docetaxel concentrations for 2D cells (8 ng/mL) was based on the minimum effective concentration established in our previous optimization studies (Essa et al., 2025). This was defined as the minimum dose required for a biological response of a 50% reduction of cell viability in PSMA-positive LnCap cells. B selecting this dose for both LNCap and PC-3 lines, we aimed to isolate the efficacy provided by the targeted delivery vehicle from the baseline cytotoxicity of the free drug. The selection criterion of the effective concentration of 20 ng/mL for the spheroid model was the same, based on the viability results in Figure 8. The free docetaxel treatment was inactive in PC-3 cells, which is likely due to the resistance of this androgen-independent cell line to low-dose taxane therapy (Liu et al., 2013).

Notably, the minimum effective concentration of the ATN treatment for spheroids is greater than that for the 2D cells (20 ng/mL for spheroids compared to 8 ng/mL for 2D LnCap cells). This could be due to the inadequate penetration of the treatments through the barrier of the extracellular matrix and dense interstitial fluid characteristic of the 3D spheroid structure (Brüningk et al., 2020), and also explains the lack of efficacy of free docetaxel on the spheroids shown in Figure 8 (80%–90% viability at all treatment concentrations) compared to LnCap 2D cells (50% viability at 28 ng/mL of free docetaxel), (Essa et al., 2025). This is consistent with the research of Xu’s group that required twice the concentration of docetaxel treatments to cause spheroid viability from 80% to 60% (Xu et al., 2022). The fact that the targeted system achieved significant cell death, migration inhibition and G2/M arrest at the concentrations that free docetaxel was ineffective highlights the therapeutic gap that the targeted system was designed for. The enhanced efficacy of the targeted system on the spheroids implies that it potentially improves the solubility and penetration of within the complex 3D microenvironment.

4.6 Inhibition of spheroid growth by targeted nanosystem

These results correlate with the relative reduction in viability between the treatments, shown in Figure 9, and demonstrates the ability of the targeted system to penetrate the 3D spheroids more efficiently than either the NTN or docetaxel. It also corresponds with the viability measurements in Figure 8, which shows no significant difference between the 20 ng/mL docetaxel treatment and the control. Enhanced targeting efficiency was reflected in the earlier and more robust growth inhibition observed with the ATN system. Moreover, both nano-systems demonstrated higher efficacy in spheroid growth suppression compared to free docetaxel starting at day 13. Additionally, Figure 9 shows morphology changes and necrosis, by the darkening of the ATN and NTN treated spheroids from days 7 and 11 respectively. This is earlier than the inhibition observations (days 11 and 13 respectively), indicating toxicity at the cellular level that occurs before changes to spheroid size.

As with in vivo studies, the relevant dosage of treatments should be optimized for 3D culture models (Friedrich et al., 2009). In previous studies (Bäcker et al., 2016; Eder et al., 2016; Jouberton et al., 2022; Raitanen et al., 2023), cell viability studies were conducted on spheroids with ranges of concentrations in order to determine the effective inhibitory concentrations of treatments for 3D multicellular tumour spheroids inhibitory concentrations (Vinci et al., 2012). Much like 2D culture measurements, the results of cell viability assays vary greatly between studies, and there have been reports of inhibitory concentrations of docetaxel on LNCap spheroids between 70 nM (56.6 ng/mL) (Edmondson et al., 2016) and 25 µM (Jouberton et al., 2022). These concentrations are much higher than what was used in this study (20 ng/mL), and it could be possible that this concentration was too low for docetaxel alone to have an effect on spheroid growth and health.

Karendish and co-workers reported on docetaxel loaded polymersomes targeted with folic acid and showed a decrease in cell viability of 88%–55% between their non-targeted and targeted systems on LNCap spheroids (Karandish et al., 2016). They tested a lower effective docetaxel concentration of 10 nM (8.1 ng/mL), while in this study there was no significant difference between the NTN and ATN treatments at 10 ng/mL. However, at an effective concentration of 20 ng/mL, the cell viability of the spheroids was reduced from 69.5% ± 6.0% to 43.4% ± 6.4%, indicating a similar improvement of the targeted system.

4.7 Applicability of cell death mechanisms and visualisation of penetration of targeted nanoparticles in 3D spheroids

In order to explore the mechanisms of cell death within the spheroid structure, the effect on intracellular processes from intact spheroids should be determined. However, a limitation of this study was the analysis of these mechanisms in the 3D format. We encountered difficulty preparing viable single cell suspensions from the whole spheroids and the 2D cell migratory assay was not compatible with the 3D format. Therefore, we investigated the induction of these mechanisms by targeted nanoparticles in the 2D format and normalised the results against the activity on PC-3 cells, which does not express PSMA receptors, as well as with NTN, the non-targeted nanoparticles. A qualitative analysis of cell migration and invasion in spheroids was described by Massaro’s group, who observed a decrease of cellular expansion in the outer proliferative layer of melanoma spheroids (Massaro et al., 2017). In our study, the outer layer of the treated LNCap spheroids showed increased cell death, contributing to the inhibition of spheroid expansion in Figure 9. The study by Massaro also described cell cycle arrest in the G2/M phase of 2D SK-Mel melanoma cell lines which is consistent with our result of nanoparticle induced G2/M cell cycle arrest in 2D LNCap cells. In our work, the difference between the targeted and non-targeted systems (observed in the PSMA-positive LNCap cells) on G2/M phase arrest and 3D spheroid growth inhibition points to a targeting-effect. In contrast, the increases in apoptosis and ROS generation, caused by both nano-systems, appear to be more characteristic of a general nanoparticle-formulation effect.

The biological significance of the targeted nanoparticle treatment on 3D spheroids is evidenced here by the reduction in cell viability and the nanoparticle penetration through the spheroid. The enhanced efficacy of the ATN system correlate with the reduction in cell viability (but no significant reduction of viability in the NTN or free docetaxel treatments) observed earlier. This observation is also consistent with the work of Karendish’s group, who observed only 55% spheroid viability in their targeted treatment group, and >86% cell viability in other treatment groups (Karandish et al., 2016).

The presence of fluorescently tagged ATN’s in the spheroids implies that the nanoparticles would have had access to the binding sites in order to enter the cells, and release docetaxel, therefore causing enhanced cell death in this treatment group. This efficient distribution of nanoparticles throughout the 3D structure of the spheroid could be due to the interaction of the targeting ligand on the nanoparticle surface with the PSMA receptors on the surface of the cells in the spheroid. The penetration of the nanoparticles could also be enhanced by the presence of the PEG groups on the PLGA core, which has been reported to reduce electrostatic interactions with the extracellular matrix (Tomasetti et al., 2016) (62), and thereby could increase the diffusion of the nanoparticles through the spheroid. This effect has also been reported by Tchoryk and co-workers who observed efficient penetration of pegylated poly (glycerol adipate) nanoparticles into the core of HCT116 colorectal cancer spheroids (Tchoryk et al., 2019).

5 Conclusions and future perspectives

In this study, optimized functionalised PLGA-PEG nanosystem was assessed for its in vitro effect on prostate cancer cell mechanisms and the inhibition of LNCap spheroid growth and viability, as well as its interaction with the 3D spheroid structure. The targeted nanosystem preferentially inhibited cell migration and cell cycle progression on LNCap cells compared to PC-3 cells. It also showed significantly higher inhibition on 3D LNCap spheroid growth, compared to free docetaxel and the non-targeted system, and demonstrated the ability to penetrate the 3D structure of LNCap spheroids. It warrants further investigation to determine if the nanoparticles can be visualized on fluorescently labelled PSMA receptors in the 3D spheroid, which would confirm the mechanism for increased cellular uptake and efficacy. The mechanisms of inhibition of cell migration and cell cycle arrest could also be investigated and compared in the spheroid model, using advanced techniques such as light sheet microscopy. The results of this work suggest that the system could be developed for in vivo studies and further preclinical investigations to assess its efficacy as a targeted treatment for prostate cancer.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Ethics statement

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.

Author contributions

DE: Methodology, Writing – original draft, Formal Analysis, Investigation, Conceptualization. MK: Writing – review and editing, Methodology, Conceptualization, Supervision, Formal Analysis. PK: Methodology, Supervision, Conceptualization, Project administration, Resources, Writing – review and editing, Formal Analysis. YEC: Project administration, Funding acquisition, Resources, Supervision, Writing – review and editing, Methodology, Conceptualization.

Funding

The author(s) declared that financial support was received for this work and/or its publication. The authors acknowledge the financial support of the National Research Foundation (NRF) of South Africa under the SARChI Chair program (grant no. PPNT230823145247) awarded to YEC.

Acknowledgements

The authors would like to thank Chris Power for assisting with the use of the confocal microscope.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The authors MK, PK, YEC declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnano.2026.1757740/full#supplementary-material

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