PSMA-Targeted Liposomes for Therapeutic Delivery into a Prostate Cancer Cell Model

Miguel Ferreira; Neelum Aziz Yousafzai; Lital Ben Naim; Bahar Ataeinia; Umar Mahmood; Pedram Heidari

doi:10.20944/preprints202607.0464.v1

Submitted:

05 July 2026

Posted:

07 July 2026

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Abstract

Background: Prostate cancer (PCa) remains a significant health challenge, requiring innovative treatments. Here, we evaluated the efficacy of Talazoparib-loaded liposomes incorporating a targeting moiety, in combination with BI 2536, in PCa. This study develops lipid nanoparticles (LNPs) targeting prostate-specific membrane antigen (PSMA) for better drug delivery. Methods: Two new PSMA-targeting agents, DUPA-DSPE and DUPA-PEG-DSPE, were created and incorporated into LNPs via thin-film hydration. LNPs’ size and morphology were characterized using Dynamic light scattering, and structural analysis was done via TEM imaging. In vitro cytotoxicity and cell viability were assessed using an MTT assay, while in vivo radioactivity was evaluated by PET imaging, and radioactive activity was quantified using a γ-counter. Results: These LNPs demonstrated excellent stability and specific binding to PSMA-expressing prostate cancer cells in vitro. In vivo, mouse model studies showed that PSMA-targeted LNPs accumulated preferentially at tumor sites. Talazoparib, a PARP inhibitor with low solubility and cytotoxicity, has limited use in PCa. Conclusions: Loading Talazoparib into DUPA-PEG LNPs significantly improved its therapeutic effect, especially when combined with BI2536. PSMA-targeted LNPs enhanced drug delivery and treatment efficacy for PCa. Further research is needed to optimize formulations and explore additional combination therapies for clinical use. This work highlights the potential of targeted delivery systems to advance PCa treatments.

Keywords:

prostate cancer

;

PSMA targeting

;

lipid nanoparticles (LNPs)

;

Talazoparib

;

combination therapy

;

drug delivery

;

preclinical evaluation

Subject:

Medicine and Pharmacology - Oncology and Oncogenics

Introduction

Prostate cancer (PCa) poses significant health and socioeconomic burdens [1]. The National Cancer Institute anticipates a 27% rise in PCa diagnosis and treatment costs in the United States, from $21.4 billion in 2020 to $27.2 billion by 2030 [2]. Early-stage PCa is typically managed through radical prostatectomy (RP) or external radiotherapy (RT). At the same time, advanced or metastatic cases may require chemical castration, often progressing to metastatic castration-resistant prostate cancer (mCRPC), characterized by high aggressiveness and mortality rates. Treatment modalities are typically tailored to the stage of the disease, the clinical context, and the availability of treatment options. Active surveillance (AS), RP, or RT are initial management options for low-risk patients [3]. Although AS may seem cost-effective due to the potential for metastatic progression, RP and RT show marginal decreases in survival rates, attributed to delayed or prevented treatment complications. In intermediate to high-risk PCa, RT combined with androgen deprivation therapy (ADT) and RP emerges as a cost-effective option, with RT particularly beneficial for older patients [4,5].

Precise tumor targeting aimed at specific cancer biomarkers for effective drug delivery potentially minimizes the side effects associated with normal tissue damage. In PCa, the highly expressed transmembrane glycoprotein prostate-specific membrane antigen (PSMA) is a crucial target for diagnosis and treatment [6]. PSMA exhibits exceptional tissue specificity to PCa, especially in poorly differentiated, metastatic, and castration-resistant tissues, making it an indispensable tool for PCa management [7,8]. Utilizing PSMA-targeted small molecules such as PSMA-I&T, PSMA-1007, PSMA-617, PSMA-11, DCF-PyL, and rhPSMA-7.3 has shown promising results in PCa imaging and therapy due to their low molecular weight, tissue penetration, rapid blood clearance, and scalability [9,10].

Nanoplatforms (NPs), including metal nanoparticles, polymer-drug conjugates, micelles, liposomes, and dendrimers, offer advantages over conventional formulations by increasing drug solubility, mitigating cytotoxicity, and improving drug pharmacokinetics [11,12]. The creation of NPs, combining drugs with molecular probes, increases the drug half-life in the circulatory system and, specifically, delivers anticancer drugs to target tissues, controlling the drug release through detectors responsive to different stimuli such as pH, temperature, light, ultrasound, and enzymatic activities, thus improving the delivering of the required drug concentration to the area of interest [13]. By means of improving the circulating half-life, nanomedicines can accumulate in tumors through the enhanced permeability and retention (EPR) effect [14]. Previous studies have reported that EPR varies among mouse models and patients, between tumor types of the same origin, and among tumors and metastases of the same patient, thus explaining the heterogeneous outcomes of NPs clinical trials. To overcome this issue, efforts should focus on methods that increase specific uptake at the tumor site. One strategy is active targeting, in which nanoparticles are conjugated to targeting agents, such as molecules that bind specifically to an overexpressed receptor on target cells [15]. This ligand-receptor interaction increases the accumulation of NPs in the specific cell, inducing their internalization and drug release via receptor-mediated endocytosis [16,17].

Here, we have developed two novel PSMA-targeting liposome platforms. In vitro and in vivo testing was conducted to assess the targeting capabilities of the liposome platform. The efficacy of Talazoparib-loaded liposomes, incorporating the targeting moiety, in combination with BI 2536, was evaluated in a prostate cancer model. Talazoparib, a PARP inhibitor encapsulated in liposomes, has shown activity for the treatment of PCa [18]. Talazoparib inhibits PARP enzymatic activity and regulates DNA damage repair, hence inducing cell death. Liposome-encapsulated Talazoparib shows increased bioavailability, enhanced drug delivery to the target, and reduced side effects [19]. BI2536 is a PlK1 inhibitor that inhibits tumor growth in vitro and in vivo across various cancers, both via cell cycle arrest, with some safe clinical trials. BI2536 is often combined with other chemotherapies to enhance its efficacy [20]. While Talazoparib is not currently approved in combination with BI2536 for PCa, preclinical evidence suggests this combination is effective in PCa [21]. In this study, we investigated a PSMA-targeted liposome platform for specific delivery of Talazoparib to PCa tumors and assessed its efficacy in combination with BI2536. We believe that targeted delivery of encapsulated Talazoparib enhances drug efficacy and reduces toxicity. We hope this platform enables us to overcome the drug resistance mechanism and improve treatment outcomes.

Materials and Methods

2.1. Reagents

Reagents, including 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-Hydroxysuccinimide (NHS), Cholesterol, Triethylamine (TEA), and Trifluoroacetic Acid (TFA), were procured from Sigma-Aldrich (St. Louis, MO, USA). Talazoparib, BI 2536, and DUPA(OtBu)-OH were obtained from MedChemExpress (Monmouth Junction, NJ). Additionally, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000] (DSPE-PEG-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-NH2), and 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). All reagents and solvents were utilized without further purification.

2.2. Synthesis

Synthesis of DUPA-DSPE and DUPA-PEG-DSPE was conducted. A quantity of 15 mg of DSPE-NH2 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) was dissolved in 3 mL of dichloromethane (DCM) and 1.5 mL of methanol (MeOH). Subsequently, 0.98 equivalents of DUPA(OtBu)-OH were dissolved in 200 µL of water and added to the previous mixture. A catalytic amount of triethylamine (TEA) was introduced, and the mixture was stirred for 16 hours. The desired product was precipitated using cold diethyl ether. The solid precipitate was then dissolved in 3 mL of trifluoroacetic acid (TFA) and stirred for 3 hours. After solvent evaporation, the resulting oil was dissolved in water, frozen, and lyophilized. The final product was obtained as a white solid with a yield of 82%. (NMR) a similar procedure was employed to synthesize DUPA-PEG-DSPE. In this process, DSPE-NH2 was replaced with DSPE-PEG-NH2 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]). The resulting product was washed three times with cold diethyl ether, affording a final yield of 94%.

2.3. Liposomes Preparation

Liposomes (LNPs) were created using the thin-layer hydration method. In brief, DPPC, cholesterol, DSPE-PEG, and DSPE-MTX were dissolved in chloroform in a round-bottom flask at a ratio of 6:3:1:1. The organic solvent was evaporated at 60°C under reduced pressure to form a thin lipid film, which was left overnight under a hood to eliminate residual solvent. To produce multilamellar liposomes, the lipid film was hydrated with 5 mL of HEPES and then subjected to three cycles (every 3 minutes) of warming at 60°C in a water bath and vortexing at 700 rpm. After 30 minutes at 60°C, the LNPs were extruded through polycarbonate membranes with pore sizes of 400, 200, and 100 nm to generate liposomes with varied sizes. For labeling with 68Ga and 64Cu, 20% w/w of DSPE-PEG was replaced with DSPE-DOTA, and the same method was used to produce these liposomes. The DOTA-LIPs were resuspended in 3 µL, and 750 mL of DOTA-LIP was conjugated with 300 µL of 68Ga or 64Cu solution (4 mCi). Excess metal ions were removed via filtration using Amicon 30 KD with 3 PBS washes. Purity was verified using iTLC with saline as the eluent. To prepare Talazoparib-loaded liposomes, 5 mg (dissolved in 50 µL DMSO) was added to the organic phase containing all lipids. The remaining steps followed the previous protocol, and any excess Talazoparib was removed through dialysis against PBS.

2.4. LNPs Characterization

Dynamic light scattering (DLS, Malvern Zetasizer Nano S) was employed to characterize LNPs’ size, morphology, and zeta potential under hydrated conditions at pH 7.0. The measurements were made with a contact angle of 173°, Backscatter at 25° C. For structural analysis via TEM imaging, liposomes were negatively stained using the following protocol: 5 µL of the sample was absorbed for 1 minute onto a carbon-coated grid (EMS, www.emsdiasum.com, order # CF400-CU) that had been made hydrophilic with a 20-second glow discharge (25 mA). The adsorption occurred in the presence of osmium vapor to stabilize the liposomes. Excess liquid was removed with filter paper (Whatman #1), and the grid was stained with 1% Uranyl Acetate (EMS catalog # 22400) for 20 seconds. Grids were examined with a TecnaiG² Spirit BioTWIN, and images recorded using an AMT NanoSprint43-MkII camera. To determine the Talazoparib encapsulation efficiency (EE), the samples were lyophilized, dissolved in acetonitrile/H2O (1:1, v/v), and analyzed by high-performance liquid chromatography (HPLC) (Agilent 1260 Infinity) with a 100 μL sample loop injector. A C18 column (2.1× 250 mm, 5 μm particle size, Agilent, USA) facilitated chromatographic separation. Talazoparib was eluted under isocratic conditions with a binary solvent system [Acetonitrile / H2O + 0.1% (v/v) TFA, 43:57 v/v], flowing at 1.0 mL/min. EE was determined using the following equation:

EE (%) = (Talazoparib weight in particles) / (Talazoparib initial feeding amount) × 100

To examine Talazoparib release kinetics, 200 μL of Talazoparib LNPs solution was placed into Slide-A-Lyzer MINI dialysis microtubes with a 10 kDa molecular cutoff (Thermo Scientific). The microtubes were dialyzed against 4 L of PBS buffer (pH 7.4). At each time point, three samples were collected, dried, and then processed. All samples were first destroyed with cold methanol, allowed to dry, and then dissolved in a mixture of acetonitrile and water (1:1, v/v). They were subsequently analyzed by HPLC to measure the MTX and Col.

2.5. In Vitro Targeting Efficacy

The PCa cell lines (22RV1, PC-3, and LNCaP) were sourced from ATCC in New York and thawed for in vitro anticancer testing. 22RV1 and LNCaP cells were grown in RPMI-1640 medium. In contrast, PC3 cells were cultured in F-12K (ATCC) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin (10000 IU/mL), and streptomycin (10000 µg/mL). The cells were maintained at 37 °C with 5% CO2. Cell viability and counts were assessed using an automated cell counter (Countess, Invitrogen) with the trypan blue exclusion method to determine live cells accurately before experiments. Cells were grown and resuspended to a density of 1 x 10⁵ cells/150 µL. After pre-wetting the 96-well Filtration Plate MultiScreen® (Millipore Sigma), 150 µL of the cell suspension was loaded into each well. The plates were incubated at 37 °C with 5% CO2 for 24 hours. Subsequently, the medium was replaced with RPMI containing the tested formulations at the respective concentrations.

2.6. In Vitro Cytotoxicity of the Liposomes

In vitro cytotoxicity was assessed for free Talazoparib, BI2546, empty liposomes, Lipo Talazoparib, and their combinations with 200mM of BI2546. Cell viability was measured using an MTT assay (Sigma-Aldrich, USA). Briefly, 22RV1 cells suspended in medium were seeded into 96-well plates at 1 × 10⁵ cells/mL and incubated at 37°C with 5% CO2 for 24 hours. The medium was then replaced with RPMI containing the respective formulations at various concentrations. After 24, 48, and 72 hours, 5.0 mg/mL MTT solution in PBS was added to each well and incubated for 4 hours at 37°C. Media and PBS were then removed, and the formazan crystals formed were dissolved in 200 μL of ethanol per well. Absorbance was measured at 570 nm with a microplate reader (Biotek Cytation 5, Agilent). Controls (cells without drug treatment) were set to 100%, and the results from treated cells were expressed as a percentage of the control viability. Each data point was replicated five times.

2.7. In Vivo Distribution of the NPs

All animal experiments received approval from our Institutional Animal Care and Use Committee (IACUC). The animals were housed in ventilated cages, with unrestricted access to food and water, in an IACUC-certified facility that provides optimal care. A total of 1.5×10^6 22RV1 cells mixed 1:1 (v:v) with Matrigel® and PBS (total volume 100µL) were injected into the left upper flank of naïve nu/nu mice. Once tumors reached a minimum volume of 800µL, mice with tumors were selected for imaging. Liposomes labeled with 64Cu, with or without PSMA tagging, were injected into five naïve nu/nu mice per group. After 24 hours, PET and CT scans were performed. Following imaging, mice were sacrificed, and their major organs (liver, lungs, heart, spleen, kidneys, intestines, and brain) were excised and analyzed for radioactive activity using a γ-counter. The activity data were normalized to the organs' weight.

2.8. Therapeutic Efficacy

1.5×10⁶ of 22RV1 cells were injected into the left shoulder of naïve nu/nu mice. The tumors were allowed to grow until reaching 100 mm³ before beginning treatment. Mice were randomly assigned to different treatment groups. In all cases, the agent was administered intravenously every 3 days for up to 21 days, for a total of 7 injections. Tumor growth was monitored using a digital caliper. All mice were euthanized when they became moribund or when tumor size exceeded 1500 mm³. Survival was tracked and analyzed using the Kaplan–Meier method.

2.9. Statistical Analysis

All statistical analyses were conducted using GraphPad Prism and SPSS. Experiments were performed in triplicate, and variables were presented as mean ± SD. The Student’s t-test and 2-way ANOVA were employed, depending on the comparison, with P values less than 0.05 regarded as statistically significant. For tumor growth curves, a linear mixed model was used to analyze tumor size data over time across different treatment groups.

Results

3.1. LNPs Preparation and Characterization

PSMA has been proven to be a valuable target for the treatment of PCa [22]. We developed two new lipid structures that can target the PSMA receptor. The final compounds, DUPA-DSPE and DUPA-PEG-DSPE (Figure 1A, B), were obtained with a yield of 84% and 92%, respectively. The liposomes (LNP) were prepared using the thin-film hydration (Bangham) method [23]. (Figure 1C, D) show a schematic representation of the two formulations prepared. As shown in the schematics, when the lipid contains a PEG linker, the formulation results in the targeting moiety being more exposed on the LNP surface. while without the PEG linker, the targeting moiety is closer to the hydrophobic lipid chains. DLS and SEM analysis show that LNPs prepared with DUPA-DSPE are 150 nm (Figure 1E, F) and those prepared with DUPA-PEG-DSPE are 120 nm (Figure 1G, H). The difference in size can be attributed to the presence and positioning of the targeting moiety within the PEG layer on the LNP surface. The DUPA structure is wider than the PEG chain, and repulsion between the carboxylic arms of DUPA and the PEG chains leads to an expansion of the PEG surface. To accommodate the DUPA moiety, the PEG surface is enlarged, resulting in a slightly larger LNP than when the DUPA is on top of the PEG surface with a PEG linker. The expansion results in slightly larger LNPs compared to formulations in which DUPA is placed on top of the PEG layer via a PEG linker.

The stability of DUPA-LNP and DUPA-PEG-LNP in PBS was assessed in comparison with non-targeted LNPs at 4°C (Figure 2A) and 37°C (Figure 2B)—over 18 days —without significant changes in LNPs' size, indicating their high stability. The targeting capability of this LNPs system was evaluated by measuring binding affinity to cells with high, moderate, and low PSMA expression (LNCaP, 22RV1, and PC3, respectively). For this aim, DOTA-DSPE and DUPA-PEG-DSPE were incorporated into the LNPs formulations, allowing radiolabeling with different radioisotopes for imaging purposes (Figures S1 and S2). No significant changes in the size or shape of the LNPs were observed. DUPA LNP, DUPA-PEG LNP, and LNP were labeled with ⁶⁸Ga due to its short half-life (T_1/2 = 68 min). (Figure 2C). Targeted nanoparticles exhibited a three-fold increase in accumulation in cells with high PSMA expression compared to cells with low PSMA expression. Non-target LNPs showed similar levels of accumulation in both high- and low-PSMA-expressing cells. Furthermore, conjugation of the targeting moiety to the lipid-PEG resulted in greater nanoparticle surface exposure, leading to a small increase in accumulation in cells with high PSMA expression. In empty wells (without cells), a small amount of residual radioactivity was detected, likely due to a limited number of LNPs retained by the well filter. At 4°C (Figure 2D), minimal internalization of the targeted and non-targeted LNPs was observed. At low temperatures, active internalization processes are inhibited. Therefore, the detected radioactivity is likely due to nonspecific interactions between the LNPs and cell-surface proteins, rather than PSMA-specific binding.

3.2. Biodistribution

To evaluate the function of LNPs formulations in vivo. For this purpose, DUPA-PEG LNPs and LNPs were labeled with ⁶⁴Cu to access biodistribution in a mouse model bearing a tumor. The LNPs were labeled with ⁶⁴Cu due to its relatively long half-life (12.7 h). Biodistribution was assessed at 24 h by PET imaging, followed by euthanasia and collection of organs for γ-counter analysis. Analysis of the PET images acquired for DUPA-PEG LNPs and non-targeted LNPs (Figure 3A) showed high accumulation in the liver, which is typical for nanoparticles with a size in the range of 120 to 150 nm, followed by lower accumulation in the thoracic cavity, corresponding to the nanoparticles present in the heart associated with the LNPs circulating in the blood pool. This occurs because of the long circulation half-life of LNPs in the blood, which a previous study reported as exceeding one day. Regarding tumor accumulation, DUPA-PEG LNPs showed higher tumor uptake than non-targeted LNPs (Figure 3B). Liver biodistribution analysis showed accumulation of 45% to 50 % ID/g of tissue for both formulations. High uptake was also observed in the spleen (93% and 73%) and kidneys (23% and 18%) for DUPA-PEG and non-targeted LNPs, respectively, consistent with LNPs pathways in mice. In addition, significant accumulation in the blood was observed at 27% and 19%, indicating longer circulation times for both formulations. Data were not significant between DUPA-PEG and non-targeted LNPs for most organs, except tumor uptake. DUPA-PEG LNPs exhibited higher tumor accumulation (20%) compared with non-targeted LNPs (11%). This higher accumulation can be attributed to the targeting moiety present on the LNPs surface. Other LNPs formulations showed accumulation like that of non-targeted LNPs, while the DUPA-PEG LNPs achieved a 2-fold increase in tumor uptake. Analyzing the tumor-to-blood ratio (Figure 3C), DUPA-PEG-LNPs showed a higher than 2.3-fold increase, whereas non-targeted LNPs exhibited only a 1.3-fold increase. Collectively, these data highlight the importance of incorporating targeting moieties into LNPs formulations to enhance tumor accumulation in mice.

3.3. Therapeutic Studies

To take advantage of the targeting moiety method, it was decided to load a Poly-ADP ribose polymerase inhibitor (PARPi) into the LNPs. PARP inhibitors have been implemented as a therapeutic choice for the treatment of PCa. Their primary mode of action is to induce synthetic lethality in cells with underlying deficiencies in homologous recombination repair (HRR). For this reason, Talazoparib® (TZPB) was loaded into the formulation. The low solubility of Talazoparib® makes it an appropriate candidate for loading into a formulation designed to increase its bioavailability at the tumor site. (Figure 4A) shows a schematic representation of DUPA-PEG LNPs loaded with TZPB. TEM analysis (Figure 4B) and DLS analysis (Figure S3) showed particles with a size of 130 nm, similar to those of LNPs not loaded with TZPB. The Encapsulation efficiency of LNPs was 85%. A stability study of the TZPB-loaded LNPs at 37°C (Figure 4C) showed size variation over 48 h, increasing from 130 nm at day 0 to 150 nm after 24 h and 197nm after 48 h, followed by a return to the original size after 72 h. This variation of size may be associated with the instability of LNPs resulting from the release of TZPB from the core. As shown in (Figure 4D), in PBS solution, an accelerated release from the LNPs was observed during the first 10 hours, during which 40% of the encapsulated drug was released. After this initial time point, a slow, steady release was observed, reaching 50% after 72 h. Under storage conditions at 4°C (Figure 4C), the LNPs exhibited size stability over time.

3.3.1. Cell Viability Studies

Using the MTT assay, the cytotoxic activity of LNPs and drugs was assessed in the 22RV1 PCa cell line at 24 h, 48 h, and 72 h (Figure 4E,F, and Supplementary Figure S4). Talazoparib concentrations ranging from 60 nM to 1 mM were tested for TZPB- and TZPB-DUPA-loaded LNPs. As shown in Figure S4, cell viability decreased in a dose-dependent manner at all evaluated time points (24 h, 48 h, and 72 h). TZPB and TZPB-DUPA LNPs exhibited IC₅₀ values of 320 and 280 µM at 72h, respectively. The cytotoxicity of empty LNPs was also assessed in 22RV1 cells, showing cell viability above 80% at all tested time points and lipid concentrations ranging from 32 nM to 50 mM. The relatively high IC₅₀ values observed for TZPB are consistent with previously reported values. Based on these results, a combination therapy, TZPB and BI2536, was investigated. These findings support the potential of combining BI2536 and TZPB for PCa treatment. In 22RV1 Cells, BI 2536 exhibited an IC₅₀ of 400 nM at 72h. For the in vitro combination study, a non-toxic concentration of BI2536 (200 nM) was selected. When TZPB and TZPB-DUPA LNPs were combined with BI 2536, high cytotoxicity was observed, resulting in low IC₅₀ values of TZPB (150 and 70 µM), respectively.

3.3.2. Therapeutic Effect on a Prostate Cancer Model

The therapeutic efficacy of lipid nanoparticles (LNPs) encapsulating TZPB was assessed using a murine 22RV1 xenograft model. (Figure 5A) represents a schematic timeline of the preclinical experiments. The study included six treatment groups, each group receiving intravenous injections every three days for 18 days (7 injections in total). The treatment groups were: free TZPB, free BI2536, TZPB-DUPA LNPs, non-targeted TZPB-LNPs combined with BI2536, TZPB-DUPA LNPs combined with BI2536, and empty LNPs (control). Therapeutic studies were conducted using 1 mM of TZPB and 400 nM of BI 2536. Tumor growth was monitored for up to 60 days after the first treatment, as shown in (Figure 5B) for all experimental groups. At baseline, no statistically significant difference in average tumor volume was observed among the six treatment groups. The DUPA-PEG LNPs, exhibiting no therapeutic effect, were included as the control group (blue line). The control group displayed steady exponential growth, beginning around day 14, when tumor volume was already approximately five times higher than at day 1. The treated group with free TZPB (orange line) showed a similar trend, although tumor growth was slower compared to the control group. The limited therapeutic effect of free TZPB in this mouse model was attributed to its low solubility and cytotoxicity. Incorporation of TZPB into DUPA-PEG LNPs (light-blue line) enhanced its therapeutic efficacy, resulting in slower tumor growth. This improvement results from the LNPs' transport properties, which increase the bioavailability of TZPB at the tumor site. However, the overall therapeutic effect remained limited by TZPB's inherently low cytotoxicity. Free BI2536 (gray line) exhibited a therapeutic effect comparable to TZPB-DUPA LNPs due to its higher cytotoxic capability than free TZPB. The combination of free BI 2536 with non-targeted TZPB-LNPs (yellow line) or TZPB-DUPA LNPs (green line) obtained the most significant therapeutic effect. Tumor growth was effectively suppressed during the treatment period, with a subsequent increase in growth rate after treatment cessation. Notably, the targeted TZPB-LNPs group showed increased tumor growth at day 21, whereas in the TZPB-DUPA LNPs group, this accelerated growth occurred after day 40. The overall survival of mice, as shown in (Figure 5C). The Kaplan-Meier survival curve indicates that mice treated with empty LNPs and free talazoparib had median survival times of 27 and 36 days, respectively, following the start of treatment. This underscores the low cytotoxicity of TZPB, which only marginally increased median survival from 18 to 24 days (Table S1). In contrast, BI 2536 and TZPB–DUPA LNPs exhibited superior efficacy, extending the median survival of mice to 42 days, and the combination of TZPB–DUPA LNPs with BI 2536 significantly prolonged mouse survival to 60 days. Encapsulation of TZPB within LNPs increased its intra-tumoral concentration, thereby enhancing cytotoxicity and improving survival outcomes.

Discussion

The use of LNPs targeting PSMA as a receptor for drug delivery to prostate cancer (PCa) is a promising approach, as demonstrated by the outstanding clinical outcomes achieved with radio-compound conjugates and synthetic PSMA-binding small molecules for imaging and treatment of prostate cancer [24,25]. Most notably, the expression of PSMA on the intercellular surfaces of some solid tumors is of interest, as it enables specific targeting of liposomes for intracellular delivery in prostate cancer [26]. LNPs have a long history as drug delivery vehicles, encapsulating various chemotherapeutic drugs as well as novel agents for PSMA-targeted therapy in PCa and other cancers [27,28]. LNPs are products of natural or synthetic polymers and exhibit specific characteristics such as drug loading, particle size, stability, and administered dose that collectively contribute to enhanced therapeutic efficacy and reduced toxicity of antitumor drugs to healthy tissues [29,30,31].

Herein, we prepared two PSMA-targeted molecules, DUPA-DSPE and DUPA-PEG-DSPE, and encapsulated the anti-cancer drugs Talazoparib and BI2536, which regulate DNA repair mechanisms and induce cell-cycle arrest in cancer cells [32,33]. The purpose of this study was to evaluate the role of novel PSMA-targeted LNP formulations in prostate cancer (PCa) using both in vitro and in vivo models and to overcome the nonspecific targeting of Talazoparib and BI2536 in 22Rv1 PCa. BI2536 is a small-molecule inhibitor of polo-like kinase 1 (PLK1), known to induce G2/M cell cycle arrest and apoptosis [21]. Previous studies have reported that PLK1 inhibition by BI2536 significantly potentiates the cytotoxic effects of PARP inhibitors, such as olaparib, in 22Rv1 cells and corresponding xenograft tumor models [34]. Yang et al, reported that DSPE-PEG LNPs conjugated with a peptide and loaded with the chemotherapeutic drug epirubicin (EPI) significantly inhibited cell proliferation and metastasis in vitro and in vivo in a breast cancer study. To assess the therapeutic effects of DP, EP, and TP as nanocarriers on breast cancer cell lines, both control and drug combination groups were evaluated. The LNPs-EPI and DP-EPI-TP groups showed significantly lower cell viability than the TP group [35]. Our results demonstrated targeted delivery of LNPs to prostate cancer (PCa) cells with low and high PSMA expression (Supplementary Figure S3). Binding affinity studies showed that targeted LNPs (DUPA-PEG LNPs) exhibited enhanced accumulation in high PSMA–expressing 22Rv1 cells compared with low PSMA–expressing PC3 cells. In contrast, non-targeted LNPs showed similar accumulation levels in both cell types. Furthermore, cell viability was assessed using targeted and non-targeted LNPs combined with therapeutic agents (Talazoparib and BI2536) at different concentrations and time points. The combined targeted LNPs significantly reduced the viability of PCa cells in vitro.

Animal studies have shown that the incorporation of TZPB into DUPA–PEG LNPs in combination with BI 2536 significantly enhanced therapeutic efficacy, resulting in slower tumor growth compared with empty targeted liposomes in a murine model of PCa. This improvement is attributed to the transport properties of LNPs, which increase the bioavailability of TZPB and BI 2536 at the tumor site. The improved median survival is associated with the higher cytotoxicity of free BI 2536 compared with free TZPB, as well as the enhanced tumor delivery of TZPB facilitated by DUPA–PEG LNPs. Moreover, the combination of TZPB–DUPA LNPs with BI 2536 further prolonged mouse survival to 60 days after treatment initiation, likely due to the synergistic interaction between TZPB and BI 2536. Encapsulation of TZPB within LNPs increased its intratumoral concentration, thereby enhancing cytotoxicity and improving survival outcomes. Notably, only the TZPB–DUPA LNP combined with BI 2536 achieved survival of nearly three months after treatment initiation, with a median survival of 81 days.

PSMA-targeted small molecule by conjugating a tumor-targeting moiety and evaluated its biodistribution in tumor tissue and healthy organs following systemic administration in tumor-bearing mice, thereby highlighting the potential benefit of this combination therapeutic modality for patients with metastatic prostate cancer [36]. The substantial improvement observed relative to non-targeted LNPs is attributed to increased TZPB accumulation mediated by the enhanced tumor-targeting capability of DUPA–PEG LNPs. While TZPB alone demonstrated limited cytotoxic activity, its combination with BI2536 resulted in a pronounced survival benefit, particularly when administered via DUPA–PEG LNPs. This enhanced efficacy is likely driven by synergistic drug interactions and improved intratumoral accumulation enabled by targeted LNP delivery. By increasing drug bioavailability while reducing off-target exposure, LNPs enhance cytotoxicity and translate these gains into meaningful survival benefits. These two novel formulations highlight the importance of nanoparticle-mediated drug delivery in maximizing the therapeutic potential of combination treatments for prostate cancer. Accordingly, LNPs represent a promising delivery platform for therapeutic agents in the treatment of advanced prostate cancer and potentially other cancers.

Conclusions

The development of lipid structures targeting the PSMA receptor represents a significant advancement in prostate cancer therapy. PSMA has been validated as a valuable therapeutic target, and the conjugation of targeting moieties such as DUPA to lipid structures enhances the specificity and efficacy of drug delivery to PSMA-expressing PCa. Formulation of lipid nanoparticles (LNPs) using thin-film hydration methods enables precise control over particle size and surface characteristics. Incorporation of PEG linkers alters the exposure of targeting moieties on the nanoparticle surface, thereby optimizing interactions with PSMA receptors and improving targeting efficacy. The synthesized LNPs exhibit excellent stability under physiological conditions and exhibit high binding affinity for PSMA-expressing cells, highlighting their potential as effective targeted drug-delivery nanocarriers. In vivo studies in a mouse model further reveal the preferential accumulation of PSMA-targeted LNPs at tumor sites, indicating their potential for targeted drug delivery. Notably, loading TZPB into DUPA-PEG LNPs enhances therapeutic efficacy, with additional improvements observed when combined with therapeutic agents.

Acknowledgements

This study was supported by NIH K08CA249047 (P.H.)

Author Contributions

Conception and study design: P.H & U.M. Methodology, experimental study, data analysis and interpretation of results, manuscripts writing: M.F, N.A.Y, L.B.N, B.A, C.L., U.M., and P.H. All authors approved the manuscript before submission.

Ethical Approval

All animal studies were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Massachusetts General Hospital, Harvard Medical School.

Competing Interests

The authors declare no competing interests. U.M. is a co-founder of CytoSite Biopharma.

Data Availability

The raw data of this study were generated at the Center for Precision Imaging, Division of Nuclear Medicine and Molecular Imaging, Department of Radiology, and Wellman Center for Photomedicine, Massachusetts General Hospital, and are available upon request to the corresponding author.

Code Availability

Not applicable.

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Figure 1. Schematic Representation of the monomers and particles prepared. Chemical structures of the two lipid-based DUPA compounds modified with DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) (A) and DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]) (B). Schematic representations and TEM image of the liposomes produced with DUPA-DSPE (C) and DUPA-PEG-DSPE (D) with scale bar of 200 nm. Hydrodynamic diameter of DUPA LPNs (E) and DUPA-PEG LNPs (F) dynamic light scattering analysis DUPA-PEG-DSPE (G, H).

Figure 2. Stability of LNPs, DUPA LNPs, and DUPA-PEG. LNPs formulations at 4°C (A) and 37°C (B). Comparison study of accumulation of DUPA-DSPE liposome, DUPA-PEG Liposome, and non-targeted liposomes into PCa cell lines (22Rv1, LNCaP, and PC3) at 37°C (C) and at 4°C (D).

Figure 3. ⁶⁴Cu imaging. PET/CT analysis of ⁶⁴Cu accumulation on subcutaneous PCa xenografts (22Rv1 cell), at 24 h post systemic administration of DUPA-PEG-LNPs vs LNPs, respectively (A). Biodistribution in mice bearing subcutaneous PCa xenografts (22RV1 cell), at 24 h post systemic administration of DUPA-PEG-LNPs vs LNPs. Data are presented as the mean ± SD; n ≥ 4 mice per experimental group (B). Tumor to blood ratio (TBR) of ⁶⁴Cu labeled DUPA-PEG-LNPs and LNPs at 24h (C).

Figure 4. Characterization and stability of liposomes with TZPB-DUPA and BI 2536. Schematic representations and TEM image of the liposomes produced with TZPB-DUPA LNPs (A). Stability of TZPB-DUPA LNPs formulation at 37°C and 4 °C (B). Talazoparib kinetics release profiles from liposomes (volume 4L); Results are expressed as the average ± SD (n=6) (C). Viability of 22Rv1 incubated with TZPB or the combination of TZPB and BI2536 (D) and the TZPB-DUPA LNPs or TZPB-DUPA LNPs in combination with BI 2536 at 72h (E).

Figure 5. Therapeutic effect on a prostate cancer model. Schematic representations of reclinical therapeutic efficacy on 22Rv1 xenograft model (A). Variation of the tumor size over time for all experimental groups (average n= 5 mice) (B); Kaplan–Meier survival curves and list of median survivals (C).

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