
==== Front
8213295
1156
Biomed Pharmacother
Biomed Pharmacother
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
0753-3322
1950-6007

39002444
10.1016/j.biopha.2024.117125
nihpa2011558
Article
PSMA-targeted combination brusatol and docetaxel nanotherapeutics for the treatment of prostate cancer
Adekiya Tayo Alex a
Hudson Tamaro b
Bakare Oladapo c
Ameyaw Edmund E. a
Adebayo Amusa a
Olajubutu Oluwabukunmi a
Adesina Simeon K. a*
a Department of Pharmaceutical Sciences, Howard University, Washington, DC, USA
b Cancer Center, Howard University, Washington, DC 20059, USA
c Department of Chemistry, Howard University, Washington, DC, USA
* Correspondence to: Department of Pharmaceutical Sciences, College of Pharmacy, Howard University, Washington, DC 20059, USA. simeon.adesina@howard.edu (S.K. Adesina).
31 7 2024
8 2024
14 7 2024
09 9 2024
177 117125117125
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Active targeting to cancer involves exploiting specific interactions between receptors on the surface of cancer cells and targeting moieties conjugated to the surface of vectors such that site-specific delivery is achieved. Prostate specific membrane antigen (PSMA) has proved to be an excellent target for active targeting to prostate cancer. We report the synthesis and use of a PSMA-specific ligand (Glu-NH-CO-NH-Lys) for the site-specific delivery of brusatol- and docetaxel-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles to prostate cancer. The PSMA targeting ligand covalently linked to PLGA-PEG3400 was blended with methoxyPEG-PLGA to prepare brusatol- and docetaxel-loaded nanoparticles with different surface densities of the targeting ligand. Flow cytometry was used to evaluate the impact of different surface densities of the PSMA targeting ligand in LNCaP prostate cancer cells at 15 min and 2 h. Cytotoxicity evaluations of the targeted nanoparticles reveal differences based on PSMA expression in PC-3 and LNCaP cells. In addition, levels of reactive oxygen species (ROS) were measured using the fluorescent indicator, H2DCFDA, by flow cytometry. PSMA-targeted nanoparticles loaded with docetaxel and brusatol showed increased ROS generation in LNCaP cells compared to PC-3 at different time points. Furthermore, the targeted nanoparticles were evaluated in male athymic BALB/c mice implanted with PSMA-producing LNCaP cell tumors. Evaluation of the percent relative tumor volume show that brusatol-containing nanoparticles show great promise in inhibiting tumor growth. Our data also suggest that the dual drug-loaded targeted nanoparticle platform improves the efficacy of docetaxel in male athymic BALB/c mice implanted with PSMA-producing LNCaP cell tumors.

Prostate specific membrane antigen
Nanoparticles
Combination therapy
Drug targeting
Reactive oxygen species
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pmc1. Introduction

Prostate cancer remains a significant health concern in contemporary oncology worldwide, necessitating innovative approaches for enhanced therapeutic outcomes [1,2]. Despite advancements in cancer research and treatment modalities, the complexity and heterogeneity of prostate cancer [1] demand precision in therapeutic strategies that can enhance drug delivery, improve efficacy, and mitigate off-target effects associated with traditional chemotherapy. Nanoparticle-based drug delivery systems have emerged as promising tools in the field of cancer therapy, offering targeted and controlled release of therapeutic agents [3,4]. Among the various nanoparticles, poly (lactic-co-glycolic acid) (PLGA) has gained attention due to its biocompatibility and tunable drug release properties [5,6].

Active targeting to cancer involves exploiting specific interactions between receptors on the surface of cancer cells and targeting moieties conjugated to the surface of vectors or carriers such that site-specific delivery is achieved [7]. Of the markers identified for PC, prostate specific membrane antigen (PSMA) has proved to be an excellent target for active targeting purposes [8]. PSMA is mainly expressed in the prostrate and is expressed at all stages of prostate cancer [9]. In these tissues, its expression is up to 1,000-fold higher compared to its expression in kidneys, small intestine, salivary gland and brain [8,10]. PSMA expression is upregulated in metastatic prostate cancer and internalized after ligand binding (receptor-mediated endocytosis) [8,11]. It’s internalization after ligand binding increases up to threefold in comparison to the basal internalization level [8]. While the very low expression of PSMA in other tissues may generate concerns for toxicity, it has been shown that PSMA targeting is dependent on a sustained retention in the tumor achievable by the enhanced permeability and retention effect, the mechanism of nanoparticle accumulation in tumors [8]. Recently, a Glu-urea-based PSMA-specific ligand (Glu-NH--CO-NH-Lys) with nanomolar binding affinities to PSMA and very high internalization ratios have been developed. PSMA binding and consequent internalization have been reported using the ligand bound to conjugates, radiotherapeutic agents, iron oxide nanoparticle [8,12–15].

Recently, we reported the development of an optimized nanoparticle formulation encapsulating brusatol and docetaxel [16]. We present here, the development and evaluation of PSMA-targeted, stealth, brusatol- and docetaxel-loaded PLGA-PEG nanoparticles for the treatment of advanced prostate cancer (Fig. 1). This approach is expected to inhibit tumor progression, enhance the effectiveness of anticancer therapy, and reverse chemoresistance to docetaxel therapy as a result modulation of Nrf2 by brusatol. In addition, nanoparticle coating by polyethylene glycol (PEG) confers “stealth” property after intravenous administration. PEG provides steric stabilization and allows nanoparticles to evade cells of the reticulo-endothelial system leading to prolonged circulation time. Consequently, accumulation in tumors as a consequence of the enhanced permeability and retention (EPR) effect takes place. PSMA targeting of the combination drug nanoparticle system then assures specificity to prostate cancer cells once localized in the tumor. This “multi-mechanistic” therapeutic approach that target multiple cancer progression pathways holds the potential to eliminate prostate cancer.

2. Experimental section

2.1. Materials

All reagents listed below were purchased commercially and used without further purification. Methoxy polyethylene glycol – b - poly (lactide -co- glycolide) (mPEG-PLGA, Mn ~ 3000: 36,000 Da) (lactic acid: glycolic acid 50:50), poly (lactide-co-glycolide)-N-hydroxysuccinimide endcap (PLGA-NHS, Mn 20,000–45,000 Da) (lactic acid: glycolic acid 50:50), were purchased from Polyscitech® (Akina Inc., West Lafayette, IN, USA). Brusatol was purchased from Carbosynth (San Diego, CA, USA). Docetaxel was purchased from Millipore Sigma (St. Louis, MO, USA). CyQUANT™ XTT Cell Viability Assay, Cell Event™ Caspase-3/7 Green Flow Cytometry Assay Kit, FxCycle™ PI/RNase Staining Solution, penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). RPMI 1640 and fetal bovine serum (FBS) were obtained from ATCC (Manassas, Virginia, United States). Triphosgene, DIPEA, L-glutamic acid di-tert-butyl ester hydrochloride, Nε-Z-L-lysine tert-butyl ester hydrochloride, Ammonium formate, O-(7-azabenzotriazol-1-yl)-N, N, N’, N’-tetramethyluronium hexafluorophosphate (HATU), N, N’-diisopropylethylamine (DIPEA), and Trifluoroacetic acid (TFA), were purchased from Chem Impex (Wood Dale, IL, USA). N, N-dimethylformamide (DMF), Methanol (MeOH), Dichloromethane (DCM), Acetonitrile (ACN), Hexanes, Ethyl Acetate (EtOAc), Silica gel, Palladium on carbon (Pd/C) were purchased from Sigma-Aldrich (Burlington, MA). Fmoc-PEG-CM (MW 3400) was purchased from Laysan Bio Inc (Arab, AL). Polyvinyl alcohol (PVA 99+% hydrolyzed, MW 89,000–98,000) was purchased from Millipore Sigma (St. Louis, MO, USA).

2.2. Methods

2.2.1. Synthesis of the glu-urea-lysine (Glu-NH-CO-NH-Lys-NH2) PSMA inhibitor for targeting to prostate cancer

The PSMA targeting ligand (1) was synthesized using a reported method with slight modifications [17]. Triphosgene (5.94 g, 20 mmol) was dissolved in dichloromethane (100 mL) in a round bottom flask on ice with stirring. In a separate container, L-glutamic acid di-tert-butyl ester hydrochloride (15.98 g, 54 mmol) was dissolved in 100 mL of DCM by vortexing, followed by the addition of DIPEA (20.8 mL, 120 mmol). The latter solution was added dropwise to the triphosgene solution with stirring for 3 h. The reaction flask was then taken out of the ice bath and allowed to stir for 20 minutes before the addition of Cbz-Lys-OtBu (20.12 g, 54 mmol) solution in DCM containing DIPEA (20.8 mL, 120 mmol) in one portion. The reaction solution was stirred for 1.5 h at room temperature and the solvent was removed by rotary evaporation (Scheme 1).

Ethyl acetate (150 mL) was added to dissolve and the resulting solution was transferred to a separatory funnel, washed with 2 N NaHSO4 (2×200 mL), followed by brine (1×200 mL), and dried over anhydrous sodium sulfate. The solvent was removed in vacuo to yield an oily material. The oil was purified using silica gel column chromatography and the pure product was eluted in a mobile phase consisting of 70:30 (Hexanes: Ethyl acetate). Concentration by rotary evaporation gave a colorless oily material which turned to a white waxy material when stored at −20°C (22.95 g, 36.9 mmol, 68.3 %). Deprotection of the Cbz protecting group by catalytic hydrogenation using ammonium formate (10 eq.) and palladium on carbon (Pd/C, 20 %) in ethanol for 4 h yielded the free amine. Filtration through celite followed by rotaiy evaporation afforded a waxy solid (15.76 g, 32.3 mmol, 87.5 %). ESI-MS m/z 488.2720; ([C24H45N3O7+H] + calcd. 488.3336) (Supporting Information Figure S1). 1H NMR (Supporting Information Figure S2) and C-13 NMR (Supporting Information Figure S3) confirm the synthesis of compound 1.

2.2.2. Synthesis of Fmoc-PEG3400-PSMA (2)

To a solution of Fmoc-PEG3400-COOH (4.5 g, 1.32 mmol) in anhydrous DMF (22 mL), was added HATU (0.95 eq., 0.48 g), DMAP (20 mol%, 0.032 g) and DIPEA (2 eq., 0.46 mL) at room temperature. The reaction mixture was stirred for 20 min to activate the acid. The synthesized PSMA targeting ligand (1) (6 eq., 3.88 g) dissolved in DMF (20 mL) was then added and the reaction was stirred overnight (Scheme 2). The reaction was concentrated in vacuo followed by dilution in methanol and subsequent purification by preparatory HPLC to isolate Fmoc-PEG3400-PSMA conjugate (2). Fmoc-PEG3400-PSMA (2) was characterized by matrix-assisted laser desorption/ionization (MALDI) (Supporting Information, Figure S5).

2.2.3. Deprotection of Fmoc-NH2-PEG3400-PSMA

To obtain t-butyl-deprotected Fmoc-NH2-PEG3400-PSMA (3), deprotection of the tert-butyl groups on Fmoc-PEG3400-PSMA conjugate (2) was achieved by dissolving the compound in 75 % TFA in DCM with stirring for 3 hours at room temperature. The reaction was monitored via analytical HPLC and allowed to continue until the peak of the starting material disappeared. The crude product was obtained by evaporation, purified by preparatory HPLC and dried in vacuo. To subsequently obtain Fmoc-deprotected NH2-PEG3400-PSMA (4), the Fmoc protecting group on the PEG3400 was removed in a solution of 20 % piperidine in DMF and stirred for 20 minutes (Scheme 3). The solution was then concentrated by rotary evaporation, diluted with methanol, and purified using prep HPLC. The compound (4) was recovered by drying under vacuum.

2.2.4. Synthesis of PLGA- PEG3400-PSMA (5)

NH2-PEG3400-PSMA (4) (0.7 g, 0.18 mmol) was dissolved in anhydrous DCM (12 mL) in a round bottom flask continuously flushed with nitrogen gas with stirring. To this solution was added DIPEA (5 eq., 0.16 mL) and DMAP (20 mol%) as acylation catalyst, followed by addition of poly (lactide-co-glycolide)-N-hydroxysuccinimide (PLGA-NHS) (0.5 eq., 2.58 g) dissolved in anhydrous DCM (66 mL). The reaction was allowed to continue for 24 h (Scheme 4). The reaction solution was concentrated in vacuo and purified by precipitation into cold methanol (1 ×500 mL) and subsequently in a methanol: diethyl ether (50:50) mixture (2 ×500 mL). The recovered targeted polymer was then dried in a vacuum oven for 48 h. The structure of the pure compound was confirmed by 1H NMR (Supporting information Figure S6).

2.2.5. Nanoparticle preparation and characterization

Nanoparticles were prepared by the emulsification-solvent evaporation method using a recently published optimized method for full details of fabrication and characterization [16]. To achieve different surface densities of targeting ligand, mPEG-PLGA was mixed with PLGA-PEG3400-PSMA (5) (0 %, 2 %, 5 %, 10 %, 20 %, 40 % and 100 %) to make up the polymer blend used for nanoparticle fabrication. Briefly, to prepare rhodamine-123 or bodipy®-loaded nanoparticles, rhodamine-123 (0.5 mg) or bodipy® (fluorescent dyes) were dissolved in the organic phase (2 mL) comprising DMSO (0.2 mL), followed by acetone (0.2 mL) and ethyl acetate (1.6 mL) by vortexing. The polymer blend (50 mg) was dissolved in the organic phase by vortexing, and the solution was emulsified into 12 mL of 0.5 % PVA solution in water using a Vibra-Cell™ probe sonicator (Model VC 750, Sonics and Materials, Newton, CT, USA). Evaporation of the obtained emulsion under a fume hood was carried out to evaporate the organic phase. The prepared nanoparticles were recovered by centrifugation after washing thrice with deionized water during centrifugation/redispersion cycles and lyophilized. For brusatol- and docetaxel-loaded nanoparticles, rhodamine-123 was replaced by brusatol and docetaxel in the formulation above and the nanoparticles were similarly prepared as described. Experiments were conducted in triplicate. For the preparation of blank nanoparticles, the polymer blend was dissolved in the organic phase and nanoparticles were prepared as described above.

2.2.6. Cell cultures

Cell culture experiments were carried out using LNCaP (PSA-expressing prostate cancer cell line) and PC-3 (non-PSA-expressing prostate cancer cell line) as model prostate cancer cell lines. The human prostate cancer cell lines were a gift from Dr. Tamaro Hudson of the College of Medicine, Howard University. The cells were cultured in RPMI 1640 supplemented with 10 % FBS (ATCC, Manassas, VA, USA) and 1 % penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The cells were maintained in a humid atmosphere containing 5 % CO2 at 37 °C.

2.2.6.1. Quantitative evaluation of cellular uptake of nanoparticles.

Flow cytometry was used to examine the impact of different surface densities of the PSMA targeting ligand on nanoparticles using modified reported methods [18–20]. LNCaP cells (5×105 cells/well) were seeded in 12-well culture plates. Following overnight incubation, the growth media was replaced with complete media containing 0.5 mg/mL of various formulations of rhodamine 123-loaded nanoparticles prepared with different surface densities of the targeting ligand. Control groups consisted of untargeted nanoparticles, and nanoparticles with 100 % surface coverage with PSMA-targeting ligand. After a 15 min and 2 h incubation, the cells were washed three times with cold PBS. The fluorescence intensity of the cells was measured in ice cold FACS buffer (3 % FBS) by BD FACSVerse flow cytometer (BD Biosciences) equipped with a 488 nm laser and 527/32 filter and analyzed using FlowJo software version 10.4.0 (Tree Star Inc., Ashland, OR, USA). All experiments were independently carried out in triplicate.

2.2.6.2. Evaluation of cytotoxicity of PSMA targeted brusatol- and docetaxel-loaded nanoparticles.

The in vitro cytotoxicity of PSMA targeted brusatol- and docetaxel-loaded nanoparticles on LNCaP and PC-3 cells was investigated using the CyQUANT XTT cell viability assay (Invitrogen, Waltham, MA, USA) by a modified published method [16]. LNCaP (6000 cells/well) and PC-3 (4000 cells/well) were seeded in 96-well plates and allowed to attach overnight. After 24 h incubation, cells were treated with 100 μl of culture medium containing PSMA-targeted brusatol- and docetaxel-loaded nanoparticles or brusatol + docetaxel in solution at the same concentration as the nanoparticle formulation (Docetaxel: 2.5 nM, 5 nM, 10 nM, 20 nM, 40 nM, 80 nM and 160 nM; Brusatol: 1.61 nM, 3.22 nM, 6.44 nM, 12.88 nM, 25.77 nM, 51.54 nM and 103.09 nM). The concentrations of each of the drugs used was based on the drug loading in the nanoparticle formulation determined by HPLC. Control cells were treated with culture medium only, DMSO solution in culture medium and culture medium containing blank nanoparticles at the highest concentration tested. At 24 h and 72 h post treatment, XTT assay was carried out per manufacturer protocol. Absorbance was read at a wavelength of 450 nm on a Biotek ELx808 absorbance microplate reader (Lonza, Walkersville, MD). Results are presented as percent viability normalized to controls. Data represents the mean ± SD of four replicates per concentration tested.

2.2.6.3. Determination of intracellular reactive oxygen species (ROS) concentrations.

Intracellular concentrations of reactive oxygen species (ROS) were measured using the 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) assay using a modified, published method [21]. LNCaP and PC-3 cells (2.5 × 105 cells/mL) were seeded in 6-well plates. Following 24 h incubation, the cells were treated with pure brusatol (38.66 nM), pure docetaxel (60 nM), mixture of pure drugs (38.66 nM brusatol + 60 nM docetaxel), and nanoparticle formulations containing equivalent amounts of the mixture of pure drugs for 2 h and 24 h. Control cells were incubated with media only. After the 2 h and 24 h period, cells were harvested, and washed three times with PBS. Afterwards, the cells were pelleted and resuspended in prewarmed PBS containing 10 μM H2DCFDA and incubated for 35 mins. Thereafter, propidium iodide (PI) (1 μg/mL) was added to each tube placed on ice to quench any extracellularly bound H2DCFDA, allowing the signal to be distinguished from the intracellular ROS response [22]. Data were collected using a BD FACSVerse flow cytometer (BD Biosciences) immediately after the addition of propidium iodide and analyzed using FlowJo software version 10.4.0 (Tree Star Inc., Ashland, OR, USA).

2.2.7. Tumor model

All animal studies were conducted in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Academy Press, 1996) and were approved by the Institutional Animal Care and Use Committee of Howard University under the approved protocol number IACUC-PHARM-21–01. For MTD studies, male athymic BALB/c nude mice (6- to 8-week-old) were used. For efficacy studies, LNCaP prostate cancer cells (2 × 106) suspended in 100 μL Matrigel (BD Bioscience) were implanted subcutaneously in the right dorsal flank region of male athymic BALB/c nude mice (5- to 6-week-old) (Charles River Laboratories International, Inc., Wilmington, MA). Mice were treated at 8 weeks post implantation. In all cases of animal experiments, animals exhibiting signs of acute pain or distress, were euthanized immediately.

2.2.7.1. Maximum tolerated dose studies.

The maximum tolerated dose was done by adapting published methods [23,24]. In order to investigate the maximum tolerated dose (MTD), 30 male athymic BALB/c nude mice were randomly assigned to different groups (n = 3) to minimize weight differences between groups. The animals were treated with brusatol solution, docetaxel solution, PSMA-targeted docetaxel-loaded nanoparticles containing docetaxel solution equivalents, PSMA-targeted brusatol-loaded nanoparticles containing brusatol solution equivalents, and the controls (PBS and blank-PSMA targeted nanoparticles) via tail vein injection once weekly for two weeks (Table 1). For this study, the MTD is defined as the highest dose that is tolerated by the animals and did not pose any life-threatening toxicity for the duration of the study. In this study, mortality, clinical signs, and body weights were evaluated. All the animals were euthanized after drug administration per approved IACUC protocol.

2.2.7.2. Antitumor efficacy studies in xenograft mouse models.

This was done by modifying a reported method [25,26]. For these studies, fifty (50) mice were randomized into 5 groups (n = 10/group) to minimize weight differences among the groups. It should be noted that some mice did not develop tumors and non-uniform group sizes were used for calculations to determine efficacy. Mice bearing PSMA-producing LNCaP tumors were intravenously administered a once weekly dose of one of: (i) PBS, (ii) blank nanoparticle control, (iii) combination docetaxel and brusatol-loaded nanoparticles, (iv) brusatol-loaded nanoparticles and (v) docetaxel-loaded nanoparticles, through the tail vein for a total of 2 weeks (Table 2). Tumor sizes, determined by weekly vernier caliper measurements and weekly measurements of body weights were recorded. The mice body weight and tumor volume were monitored for a total of 3 weeks after treatment. Tumor volume was calculated using the formula (cm3 = 0.52 X [length x width2 (cm2)]. Relative tumor volume (RTV) over a 21-day period was calculated by dividing the tumor volume at the end of the study by the tumor volume at initial treatment time (day 0) for each mouse using the following formula: Relative Tumor Volume=(Tumor volume on day 21)Tumor volume on day 0

Tumor-bearing mice were euthanized before the tumors attained a size of 2 cm in diameter in accordance with Howard University IACUC approved protocol. Mice were euthanized by anesthetization and carbon dioxide narcosis in their home cage at the end of the experiments.

2.2.8. Statistical analysis

In the antitumor efficacy studies, one-way Analysis of Variance (ANOVA) of the mean RTV of the different treatment groups was used to compare tumor growth rates by evaluating the statistical differences between treatment groups and controls; a p < 0.05 is statistically significant. IBM SPSS Statistics software (Version 29.0.0.0 (241), IBM Corp., Armonk, NY) was used for all statistical analyses.

3. Results

3.1. Synthesis of PSMA targeting ligand and conjugation to polymer

The synthesis of the PSMA targeting ligand has been widely reported and used for active targeting to prostate cancer [8,12–15,17,27]. Characterization by ESI-MS confirms the mass of the PSMA targeting ligand (1) (Supporting Information Figure S1). Proton NMR and C-13 NMR confirm the synthesis of the PSMA-targeting ligand (Supporting Information Figure S2 and S3 respectively). Conjugation of the PSMA targeting ligand to Fmoc-protected PEG3400 yielded Fmoc-NH2--PEG3400-PSMA (tert-butyl-protected). Characterization by MALDI confirm the structure of the compound (2) (Supporting Information Figure S5). Deprotection of the tert-butyl groups and Fmoc protecting group on (2) yielded NH2-PEG3400-PSMA (4). PLGA-NHS was reacted with (4) to yield the PSMA targeted polymer (PLGA-PEG3400-PSMA). Proton NMR confirmed the structure of the targeted polymer (5) (Supporting Information Figure S6).

3.2. Effect of PSMA targeting ligand surface density on cellular uptake of nanoparticles

Cellular internalization was evaluated by flow cytometry to study the effect of PSMA targeting and ligand density on the surface of rhodamine-123-loaded nanoparticles in the two prostate cancer cell lines. Different targeting ligand densities on the nanoparticle surface was achieved by blending different mass ratios of mPEG-PLGA with PSMA-PEG3400-PLGA (e.g. 2 % formulation is a 2–98 mass ratio of PSMA-PEG3400-PLGA to mPEG-PLGA) to obtain the polymer blend used in nanoparticle fabrication. Rhodamine-123 encapsulated PSMA-targeted nanoparticles were prepared using the reported optimized formulation using selected polymer blend compositions. The composition of the polymer blends and the nanoparticle sizes obtained are presented (Table 3):

The optimal ligand density is the minimum amount of targeting ligand on the nanoparticle surface to confer maximal targeted cellular uptake. To determine the optimal surface density of nanoparticles, statistical analysis of flow cytometry data in LNCaP prostate cancer cells using the independent-samples Kruskal-Wallis test show that there is a significant difference in uptake between the nanoparticle formulation prepared with 10 % of the targeted polymer and the untargeted nanoparticle formulation (p = 0.027) and there was no significant difference between the 10 % targeted formulations when compared with formulations containing higher percentages of the PSMA-targeted polymer after treatment of LNCaP cells with the nanoparticle formulation for 15 min (Fig. 2a). Similarly, statistical analysis of the data obtained at 2 h post treatment of LNCaP cells with targeted nanoparticles reveal a significant difference in uptake between the nanoparticle formulation prepared with 10 % of the targeted polymer and the untargeted nanoparticle formulation (p = 0.008) (Fig. 2b). Interestingly, at the 2 h time point, there was a significant difference between the 100 % targeted nanoparticle formulation and both the 10 % (p = 0.032) and 20 % (p = 0.025) targeted nanoparticle formulations with the lower percentages showing much improved uptake in the cells. As with the 15 min data, there was no significant difference between the 10 % targeted formulations when compared with formulations containing higher percentages of the PSMA-targeted polymer after treatment of LNCaP cells except for the 100 % targeted formulation. Hence, the polymer blend containing 10 % of the PLGA-PEG3400-PSMA by mass was found suitable and used for nanoparticle fabrication for further biological studies.

3.3. In vitro cytotoxicity evaluations

To evaluate the cytotoxicity of the brusatol- and docetaxel-loaded PSMA-targeted nanoparticles, LNCaP and PC-3 cells seeded in 96-well plates were treated with different concentrations of brusatol- and docetaxel-loaded nanoparticle formulation prepared with 10 % of the targeted polymer and other controls. Cytotoxicity was evaluated using the XTT assay and the percentage viability was calculated relative to untreated controls. The percent viability of LNCaP and PC-3 prostate cancer cells after treatment with different concentrations of the optimized targeted nanoparticle formulation for 24 h and 72 h is shown below (Fig. 3). The optimized drug loadings for targeted nanoparticles are 2.31 % and 0.95 % for docetaxel and brusatol, respectively.

In general, the nanoparticle formulation showed concentration-dependent toxicity against LNCaP and PC-3 cells. In addition, more cell death was observed at 72 hours compared to 24 hours in the PC-3 cell line. Furthermore, greater cell death was observed in LNCaP cells treated with drug-loaded PSMA-targeted nanoparticles compared to PC-3 cells. We had previously reported that docetaxel- and brusatol-loaded untargeted nanoparticles are more cytotoxic to LNCaP cells compared to PC-3 cells [16]. Additionally, the greater cell death observed in LNCaP may indicate an interaction between the PSMA targeting ligand on the nanoparticle surface and the PSMA receptor that is overexpressed on LNCaP cells but not on PC-3 cells. PSMA targeting facilitates receptor-mediated endocytosis of the drug-loaded nanoparticles and intracellular drug release within cancer cells which correlates with improved cytotoxicity [28]. Furthermore, blank nanoparticles showed no toxicity to cells, confirming that the observed toxicity is due to the encapsulated drugs.

3.4. ROS level determination

To evaluate the effect of the PSMA-targeted brusatol- and docetaxel-loaded nanoparticles on reactive oxygen species (ROS) levels in LNCaP and PC-3 prostate cancer cells, the cells were treated with the PSMA targeted nanoparticle formulation containing equivalent amounts of mixture of pure docetaxel and brusatol, pure brusatol, pure docetaxel and mixture of pure drugs in solution for 2 h and 24 h. Control cells were incubated with media only. Levels of reactive oxygen species at the different time points were determined using a ROS-sensitive fluorescent indicator, H2DCFDA, by flow cytometry (Fig. 4). Basal levels of ROS were detected in untreated LNCaP and PC-3 cells consistent with previous report that shows that cancer cells have high basal levels of ROS [29]. When compared to untreated cells (i.e. control and control 2), all treatments significantly increased ROS generation in both cell lines over time. This is in agreement with the literature, which shows that both docetaxel [30,31] and brusatol [32,33] promote ROS production in cancer cells. Additionally, in PSMA +ve LNCaP cells, the combination drug treated cells showed higher ROS levels when compared to cells treated with individual drugs. A significant difference in ROS levels was recorded in LNCaP cells treated with the PSMA-targeted nanoparticle formulation. However, in PSMA-ve PC-3 cells, no significant difference in ROS levels was observed in cells treated with the PSMA-targeted nanoparticle formulation when compared with ROS levels of cells treated with individual drugs. Considering that the brusatol- and docetaxel-loaded nanoparticle formulation demonstrated greater cell death in the LNCaP cell line compared to the PC-3 cell line in cytotoxicity studies, increase in ROS generation facilitated by PSMA targeting may be an additional mechanism favoring cell death in LNCaP compared to PC-3 cells.

3.5. MTD Studies

In MTD studies, there was no mortality at all administered doses for the duration of the study in any of the treatment groups. In addition, there were no abnormal clinical observations throughout the period of the experiments. The mean body weights of mice in the treatment groups were comparable to the control and blank nanoparticle groups with no significant difference from day to day between groups (p > 0.05) (Fig. 5) (Supporting Information Table S1). Thus, the decision was taken to treat mice for efficacy studies at the highest concentration of drugs tested.

3.6. In vivo efficacy studies

Efficacy studies were carried out in male athymic BALB/c mice implanted with PSMA-producing LNCaP cell tumors. The mice were randomized into five treatment groups and treated by tail vein injection. Tumor size was measured at defined intervals and the tumor volume was calculated. Using the tumor volume data, the RTV was calculated, and the data subjected to statistical analysis.

Analysis of variance (ANOVA) across the different groups was evaluated using SPSS® software. ANOVA shows no significant difference across all treatment groups (p = 0.093; α = 0.05) (Supporting Information Table 1). Post hoc tests show significant differences in tumor volume changes of mice treated with dual-drug loaded nanoparticles compared with docetaxel-loaded nanoparticles (p = 0.026); and tumor volume changes of mice treated with brusatol-loaded nanoparticles compared with docetaxel-loaded nanoparticles (p = 0.011) (Supporting Information Table S2)

The means plot of the treatment groups show that brusatol-containing nanoparticles (both PSMA-targeted brusatol-loaded nanoparticles and PSMA-targeted brusatol- and docetaxel-loaded nanoparticles) contribute significantly to the antiproliferative effect and reduction in tumor volume observed compared to the other treatment groups (Fig. 6).

4. Discussion

We have recently reported the preparation, optimization and evaluation of untargeted brusatol- and docetaxel-loaded nanoparticle formulations in vitro using PC-3 and LNCaP prostate cancer cells [16]. The data revealed that the drug combination showed synergistic cytotoxic effects. In addition, the data showed that there is no significant difference between the cytotoxicity of the optimized nanoparticle formulation and a mixture of the drug solutions at the same concentrations using the MTS assay. However, cytotoxicity studies, in addition to evaluation of caspase 3/7 activity and total death by flow cytometry suggest that the mechanisms of cytotoxicity of the drug combination differ among cell cultures. In this work, a PSMA targeting ligand was synthesized and coupled to the surface of brusatol- and docetaxel-bearing nanoparticles to evaluate the impact of active PSMA targeting on the cytotoxicity of the nanoparticle formulation.

The urea based PSMA targeting agents were synthesized in the early 2000s and have been used to target PSMA for imaging and treatment of prostate cancer. Successful urea-based, PSMA targeted, FDA approved products include 177Lu-PSMA-617 (177Lu-vipivotide tetraxetan, Pluvicto™, (AAA, Millburn, NJ, USA)), approved in the US for the treatment of metastatic castration-resistant prostate cancer in March 2022 and 68Ga-PSMA-11, approved for the positron emission tomography (PET) imaging of PSMA-positive prostate cancer [34,35]. Thus, there is clinical validation for the use of the targeting ligand in the targeted drug delivery to PSMA positive prostate cancer. PSMA binding and consequent internalization have been reported for the urea-based targeting ligand bound to conjugates, radiotherapeutic agents, iron oxide nanoparticles [8,12–15,27,36] hence, in this work, we synthesized PSMA targeting ligand-bound PLGA-PEG polymer for the fabrication of combination drug encapsulated nanoparticles. The targeting ligand was covalently linked to PEG3400 via a stable amide bond to form PEG-PSMA and this was covalently coupled to PLGA-NHS to form PLGA-PEG-PSMA. It has been reported that PLGA-PEG co-polymer form corona-core systems with the PEG corona extending into the aqueous circulation and overlaying the hydrophobic PLGA core [16,37]. Thus, using this approach, the hydrophilic PSMA targeting ligand is expected to be on the surface of the nanoparticle facilitating targeting to PSMA on the surface of prostate cancer cells.

While a lot of work has been reported on PSMA binding studies using the urea-based targeting ligands, less work has been done to determine optimal surface densities for receptor targeting. In this study, it was observed that as the PSMA targeting ligand density increased from 2 % to 40 %, the particle size increased from 185 nm to 313 nm (Table 3). Flow cytometry was used to evaluate the optimum ligand surface density of rhodamine-123 loaded, PSMA-targeted nanoparticles in PSMA +ve LNCaP cells. The optimum ligand density is the minimum amount of the targeting ligand on the nanoparticle surface to confer maximum PSMA facilitated cellular uptake [38]. The data showed that the internalization of targeted fluorescent nanoparticles increased up to 10 % targeted formulations. It has been reported that sufficient targeting ligands must be available to facilitate ligand binding and receptor-mediated endocytosis, however, ligand crowding or overcrowding which can lead to reduced binding must be avoided [39,40]. Above 10 % density, there was no significant difference in nanoparticle internalization based on the measurement of fluorescence intensity to warrant the use of higher surface densities in nanoparticle formulations. This is consistent with literature reports [38,41]. Thus, the favorable particle size (~ 200 nm) and internalization data of the 10 % targeted nanoparticle formulation led to the choice of the 10 % targeted formulation for the fabrication of brusatol- and docetaxel-loaded, PSMA-targeted nanoparticle formulations.

Evaluation of cytotoxicity in PSMA +ve LNCaP cells and PSMA −ve PC-3 cells by the XTT assay show differential cytotoxicity of the PSMA-targeted nanoparticle formulation in cell cultures. The targeted formulation was more cytotoxic to PSMA +ve LNCaP cells compared to PSMA −ve PC-3 cells at both 24 h and 72 h (Fig. 3). While this may suggest the importance of PSMA targeting, our previous experience shows this may not be the only case. We have previously shown that the mechanisms of toxicity and cell death differ in the cell lines and confirmed that LNCaP cells are more sensitive to untargeted brusatol- and docetaxel-loaded nanoparticle formulations compared to PC-3 cells [16]. An interesting observation is that the time-dependent cytotoxicity observed in PC-3 cells was not observed in LNCaP cells treated with the targeted nanoparticle formulation for 24 h and 72 h. This is also in contrast to our previous data which clearly shows time-dependent cytotoxicity when LNCaP cells were treated with untargeted dual drug-loaded nanoparticles [16]. This different outcome may be adduced to the rapid cellular internalization of the targeted nanoparticles as a result of PSMA binding, resulting in rapid and more efficient cell death. Uptake studies (15 min and 2 h) using flow cytometry confirms the rapid uptake of targeted particles compared to untargeted particles.

Reactive oxygen species refers to unstable and highly reactive oxygen derivatives which include hydrogen peroxide, superoxide anion, singlet oxygen, hydroxyl radical etc. which normally act as second messengers in cellular signaling pathways and are important for the proper functioning of different cellular processes [29,42]. It has been reported that most chemotherapeutic agents increase the intracellular levels of ROS in cancer cells as a result of mitochondria ROS generation and inhibition of the cellular antioxidant systems. These mechanisms increase ROS levels in cancer cells which already have high ROS as a result of increased metabolic rate, gene mutation, and relative hypoxia, leading to oxidative stress which ultimately leads to cell death [29,42,43]. Thus, the combination of chemotherapeutic agents to induce the production of ROS selectively in cancer cells along with other mechanisms may be an effective strategy to improve therapeutic efficacy in the treatment of cancers. Several reports have shown that brusatol increases ROS levels by inhibiting the antioxidant response [31–33]. Similarly, docetaxel has been reported to increase ROS levels by ROS generation [30,31]. Thus, the combination of both agents in a nanoparticle platform is hypothesized to increase ROS levels selectively in cancer cells as a result of PSMA targeting leading to the augmentation of cell death mechanisms. Our data shows a significant increase in % ROS levels in LNCaP cells treated with the PSMA targeted nanoparticles when compared with solutions of individual drugs at the same concentrations for 2 h and 24 h. This observation supports increased uptake facilitated by PSMA targeting in the PSMA +ve cell line. For PC-3 cells treated with the same nanoparticle concentrations, such increase was not observed which could be as a result of limited internalization. The combination drug solution facilitates uptake and accumulation in the cell by passive diffusion leading to increased ROS levels. On the other hand, uptake and accumulation of the PSMA targeted nanoparticles is hindered compared to free drugs because PC-3 cells are PSMA negative and is not able to facilitate uptake by receptor-mediated endocytosis. The differences in the rate of nanoparticle internalization by PSMA +ve LNCaP compared with PSMA −ve PC-3 cells is postulated to be responsible for the difference observed in percent ROS levels. We have previously reported the effect of time on the uptake of untargeted polymeric nanoparticles [44].

To evaluate the drugability and toxicity of the PSMA-targeted nanoparticle formulation in xenograft models of prostate cancer, studies were first carried out to determine the maximum tolerated dose (MTD) of the drugs to guide the choice of the dose to be administered in subsequent efficacy studies. The end points evaluated were mortality, clinical signs, and body weights. In this study, no mortality was observed for the duration of the experiment. In addition, there were no abnormal clinical observations throughout the period of the experiment. Furthermore, the body weights of mice in the treatment groups were comparable with the control group and no clear trend could be established in weight changes; thus, the highest administered dose was adjudged safe and selected for efficacy studies in mice.

To evaluate the antitumor effect of PSMA targeted drug-loaded nanoparticles, tumor volumes were calculated using the modified ellipsoidal formula i.e. Tumor Volume = 0.52 x (L x W2) [45–47] after non-invasive tumor length and width measurements with calipers. Several mice in the different groups did not show palpable tumors and were excluded from the efficacy studies. Evaluation of antitumor efficacy was done by calculating the relative change in tumor volume from baseline and subjecting the data to statistical analysis [48].

The study compares five treatment groups, including PBS, PSMA-targeted blank nanoparticles (blank), PSMA-targeted brusatol-loaded nanoparticles (brusatol), PSMA-targeted docetaxel-loaded nanoparticles (docetaxel), and PSMA-targeted brusatol- and docetaxel-loaded nanoparticles (dual), with sample sizes ranging from 6 to 8. Mean %RTV values range from 83.78 (brusatol) to 154.24 (docetaxel), indicating varying levels of treatment effectiveness. Standard deviations and confidence intervals for mean %RTV across treatments suggest differences in variability and precision of the estimates. For instance, the docetaxel group shows a large standard deviation, highlighting considerable variability within this treatment group. The ANOVA test, with an F-statistic of 2.211 and a p-value of 0.093, suggests no significant differences between the groups at the conventional alpha level of 0.05. However, the p-value is close to the threshold, indicating a trend that might warrant further investigation. The Welch (p = 0.186) and Brown-Forsythe (p = 0.108) tests offer a more robust analysis of mean differences when assumptions of homogeneity of variances are violated. Neither test shows significant differences across treatments, supporting the ANOVA findings. Post hoc tests such as the least significant difference (LSD) method identified significant mean differences between certain treatment pairs e.g. brusatol and docetaxel (p = 0.011), dual and docetaxel (p = 0.026). These results show that %RTV significantly increases with docetaxel treatment compared to brusatol treatment. Similarly, %RTV significantly increases with docetaxel treatment compared to treatment with the dual drug-loaded nanoparticles. These findings highlight specific treatment effects within the overall analysis framework. In addition, a plot of mean %RTV values visually confirms docetaxel as having the highest positive mean change in tumor volume, suggesting it might be the least effective treatment among those tested. This plot also aids in visually assessing the spread and central tendency of %RTV across treatments.

Overall, the analysis indicates that while ANOVA did not find significant differences across all treatments, specific pairwise comparisons did reveal significant effects. The significant variability within groups, particularly for docetaxel, and the close p-value from ANOVA suggest that the effect of treatments on %RTV is nuanced, with certain treatments potentially offering substantial benefits over others. These results underscore the complexity of treatment effects and the importance of considering both statistical significance and effect sizes, alongside robust testing methods, to fully understand treatment efficacy. Future research might focus on larger sample sizes or additional variables to further elucidate these effects.

5. Conclusion

Synthesis and characterization of the PSMA-targeting ligand was successfully done and coupled to amine-terminated PEG3400-COOH followed by conjugation to PLGA-NHS to yield PSMA-targeted PLGA-PEG copolymer. This copolymer was blended with different ratios of untargeted PEG-PLGA and used for nanoparticle fabrication yielding different degrees of nanoparticle surface conjugation. Evaluation of targeting ligand surface density on cellular uptake confirm a 10 % optimal targeted polymer blend. The data also shows an increase in particle size with increase in percent surface conjugation to the PSMA targeting ligand with deviation at 100 % coverage. Comparative analysis between untargeted nanoparticles and PSMA-targeted nanoparticles showed that PSMA targeting improved the cytotoxicity of docetaxel- and brusatol-loaded nanoparticles in PSMA +ve LNCaP cells but not in PSMA −ve PC-3 cells. Similarly, increased ROS generation in LNCaP cells may contribute to higher cytotoxicity in addition to improved uptake via PSMA targeting relative to PC-3 cells. Studies in PSMA-producing LNCaP xenografts show that brusatol-containing nanoparticles may contribute significantly to the antiproliferative effect of the dual drug nanoparticles. The analysis suggests that the tumor volume increased with docetaxel treatment compared to other treatments. However, the overall ANOVA result was not statistically significant at the 0.05 level, indicating that while there are differences observed, these might not be strong enough to conclusively determine the effect of treatments across all groups without further study. The violation of homogeneity of variances also calls for caution in interpreting these results, suggesting that alternative statistical designs might be needed for a more definitive conclusion. Based on our data, the synergistic effect of this PSMA-targeted combination is worthy of further consideration in the treatment of advanced prostate cancer.

Supplementary Material

Supplementary Material

Acknowledgements

We are grateful to Mr. Bell of Veterinary Services at Howard University for assistance with animal experiments.

Funding

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number SC1GM131982. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Declaration of Competing Interest

The authors declare the following financial interests: SKA reports that funding for the work was provided by NIGMS/NIH, USA. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Fig. 1. Schematic of actively targeted, corona-core drug-loaded nanoparticle. The core of the nanoparticle is made up of poly (lactide-co-glycolide) (PLGA), the corona consist of polyethylene glycol (PEG) and the targeting ligand is the PSMA binding ligand.

Fig. 2. Statistical analysis of flow cytometry data of rhodamine-123 loaded nanoparticles fabricated with different blends of PLGA-PEG-PSMA: PLGA-PEG at different time periods (a) 15 mins, (b) 2 h.

Fig. 3. In vitro percent cell viability of LNCaP and PC-3 cells treated with docetaxel- and brusatol-loaded PSMA-targeted nanoparticles and controls at different concentrations (a) LNCaP cells (24 hours), (b) PC-3 cells (24 hours), (c) LNCaP cells (72 hours), (d) PC-3 cells (72 hours). Data are mean ± SD (n=4).

Fig. 4. Determination of reactive oxygen species (ROS) in unstimulated control (control - cells incubated with media only), control 2 (cells incubated with ROS-sensitive fluorescent indicator (H2DCFDA), pure brusatol (38.66 nM), pure docetaxel (60 nM), mixture of pure drugs (38.66 nM brusatol + 60 nM docetaxel), and nanoparticle formulations containing equivalents of the pure drug mixture at 2 h, and 24 h in LNCaP cells (left) and PC-3 cells (right).

Fig. 5. Mean body weight-time profile of mice treated with brusatol (5 mg/kg and 10 mg/kg), docetaxel (13.5 mg/kg and 27 mg/kg), PSMA-targeted docetaxel-loaded nanoparticles (13.5 mg/kg and 27 mg/kg), PSMA-targeted brusatol-loaded nanoparticles (5 mg/kg and 10 mg/kg), and the controls (PBS and blank-PSMA targeted-nanoparticles) following tail vein injection (n = 3).

Fig. 6. Mean plot of relative tumor volumes.

Scheme 1. Synthesis of Glu-NH-CO-NH-Lys-NH2 (1) (PSMA targeting ligand).

Scheme 2. Synthesis of Fmoc-PEG3400-PSMA.

Scheme 3. tert-butyl and Fmoc deprotection.

Scheme 4. Synthesis of PLGA-PEG3400-PSMA (5).

Table 1 Mice treatment groups and dose administered.

S/N	Groups	Dose	
1	PBS Control		
2	PSMA-targeted blank nanoparticles	Same amount as in highest concentration of nanoparticles	
3	Brusatol	5 mg/kg	
4	Brusatol	10 mg/kg	
5	Docetaxel	13.5 mg/kg	
6	Docetaxel	27 mg/kg	
7	PSMA-targeted docetaxel-loaded nanoparticles (docetaxel equivalents)	13.5 mg/kg	
8	PSMA-targeted docetaxel-loaded nanoparticles (docetaxel equivalents)	27 mg/kg	
9	PSMA-targeted brusatol-loaded nanoparticles (brusatol equivalents)	5 mg/kg	
 10	PSMA-targeted brusatol-loaded nanoparticles (brusatol equivalents)	10 mg/kg	

Table 2 Mouse groups and treatment administered in efficacy studies.

S/N	Groups	Dose	
1	PBS (control)	100 μL	
2	PSMA targeted blank nanoparticles	Same concentration as combination nanoparticles	
3	PSMA-targeted combination docetaxel- and brusatol- loaded-nanoparticles	27 mg/kg (doc); 11.2 mg/kg (brusatol)	
4	PSMA-targeted docetaxel-loaded nanoparticles	27 mg/kg (docetaxel equivalents)	
5	PSMA-targeted brusatol-loaded nanoparticles	11.2 mg/kg (brusatol equivalents)	

Table 3 Fluorescent dye (rhodamine-123)-loaded PSMA-targeted PEG-PLGA nanoparticle composition and particle size.

Formulations (% ligand density)	PLGA-PEG-PSMA (mg)	PLGA-PEG (mg)	Rhodamine (mg)	Size (nm)	
0 % dye Formulation	10	40	-	228.8±2.0	
2 % Formulation	 1	49	0.5	185.4±2.3	
5 % Formulation	 2.5	47.5	0.5	199.9±5.9	
10 % Formulation	 5	45	0.5	209.4±2.7	
20 % Formulation	10	40	0.5	258.2±6.7	
40 % Formulation	20	30	0.5	313.0±4.7	
100 % Formulation	50	 0	0.5	224.6±7.8	

CRediT authorship contribution statement

Edmund E. Ameyaw: Formal analysis. Oladapo Bakare: Writing – review & editing, Supervision, Funding acquisition, Formal analysis. Tamaro Hudson: Methodology, Investigation, Funding acquisition. Tayo Alex Adekiya: Writing – original draft, Methodology, Investigation, Data curation. Simeon Kolawole Adesina: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. Olajubutu Oluwabukunmi: Writing – review & editing, Data curation. Amusa Adebayo: Writing – review & editing, Supervision, Investigation.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biopha.2024.117125.
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References

[1] Assadi M , Jokar N , Ghasemi M , Nabipour I , Gholamrezanezhad A , Ahmadzadehfar H , Precision medicine approach in prostate cancer, Curr. Pharm. Des 26 (31 ) (2020) 3783–3798, 10.2174/1381612826666200218104921.32067601
[2] Gillessen S , Attard G , Beer TM , Beltran H , Bjartell A , Bossi A , Briganti A , Bristow RG , Chi KN , Clarke N , Davis ID , Management of patients with advanced prostate cancer: report of the advanced prostate cancer consensus conference 2019, Eur. Urol 77 (4 ) (2020) 508–547, 10.1016/j.eururo.2020.01.012.32001144
[3] Sahu T , Ratre YK , Chauhan S , Bhaskar LV , Nair MP , Verma HK , Nanotechnology based drug delivery system: current strategies and emerging therapeutic potential for medical science, J. Drug Del. Sci. Technol 63 (2021) 102487, 10.1016/j.jddst.2021.102487.
[4] Sharma P , Mehta M , Dhanjal DS , Kaur S , Gupta G , Singh H , Thangavelu L , Rajeshkumar S , Tambuwala M , Bakshi HA , Chellappan DK , Emerging trends in the novel drug delivery approaches for the treatment of lung cancer, Chem. -Biol. Interact 309 (2019) 108720, 10.1016/j.cbi.2019.06.033.31226287
[5] Lu Y , Cheng D , Niu B , Wang X , Wu X , Wang A , Properties of poly (lactic-co-glycolic acid) and progress of poly (lactic-co-glycolic acid)-based biodegradable materials in biomedical research, Pharmaceuticals 6 (3 ) (2023) 454, 10.3390/ph16030454.
[6] Zeb A , Gul M , Nguyen TT , Maeng HJ , Controlled release and targeted drug delivery with poly (lactic-co-glycolic acid) nanoparticles: reviewing two decades of research, J. Pharm. Investig 52 (6 ) (2022) 683–724, 10.1007/s40005-022-00584-w.
[7] Liang C , Yang Y , Ling Y , Huang Y , Li T , Li X , Improved therapeutic effect of folate-decorated PLGA-PEG nanoparticles for endometrial carcinoma, Bioorg. Med. Chem 19 (2011) 4057–4066, 10.1016/j.bmc.2011.05.016.21641806
[8] Wüstemann T , Bauder-Wüst U , Schäfer M , Eder M , Benesova M , Leotta K , , Design of internalizing PSMA-specific glu-ureido-based radiotherapeuticals, Theranostics 6 (8 ) (2016) 1085–1095, 10.7150/thno.13448.27279903
[9] Afshar-Oromieh A , Avtzi E , Giesel F , Holland-Letz T , Linhart H , Eder M , Eisenhut M , Boxler S , Hadachik BA , Kratochwil C , Weichert W , Kopka K , Debus J , Haberkorn U , The diagnostic value of PET/CT imaging with the 68Ga-labelled PSMA ligand HBED-CC in the diagnosis of recurrent prostate cancer, Eur. J. Nucl. Med. Mol. Imag 42 (2 ) (2015) 197–209, 10.1007/s00259-014-2949-6.
[10] Dewes S , Schiller K , Sauter K , Eiber M , Maurer T , Schwaiger M , Gschwend JE , Combs SE , Habl G , Integration of 68Ga-PSMA-PET imaging in planning of primary definitive radiotherapy in prostate cancer: a retrospective study, Radiat. Oncol 11 (1 ) (2016) 1–8, 10.1186/s13014-016-0646-2.26743131
[11] Bouchelouche K , Choyke P , Capala J , Prostate specific membrane antigen - a target for imaging and therapy with radionuclides, Discov. Med 9 (44 ) (2010) 55–61.20102687
[12] Eder M , Schäfer M , Bauder-Wüst U , Hull W , Wängler C , Mier W , Haberkorn U , Eisenhut M , 68 Ga-complex lipophilicity and the targeting property of a urea-based PSMA inhibitor for PET imaging, Bioconjugate Chem. 23 (4 ) (2012) 688–697, 10.1021/bc200279b.
[13] Azad B , Banerjee S , Pullambhatla M , Lacerda S , Foss C , Wang Y , Ivkov R , Pomper MG , Evaluation of a PSMA-targeted BNF nanoparticle construct, Nanoscale 7 (10 ) (2015) 4432–4442, 10.1039/c4nr06069e.25675333
[14] Banerjee S , Chen Z , Pullambhatla M , Lisok A , Chen J , Mease R , Pomper M , Preclinical comparative study of 68Ga-labeled DOTA, NOTA, and HBED-CC chelated radiotracers for targeting PSMA, Bioconjugate Chem. 27 (6 ) (2016) 1447–1455, 10.1021/acs.bioconjchem.5b00679.
[15] Benesova M , Schafer M , Bauder-Wust U , Afshar-Oromieh A , Kratochwil C , Mier W , Haberkorn U , Kopka K , Eder M , Preclinical evaluation of a tailor-made DOTA-conjugated PSMA inhibitor with optimized linker moiety for imaging and endoradiotherapy of prostate cancer, J. Nucl. Med 56 (6 ) (2015) 914–920, 10.2967/jnumed.114.147413.25883127
[16] Adekiya TA , Moore M , Thomas M , Lake G , Hudson T , Adesina SK , Preparation, optimization, and in-vitro evaluation of brusatol-and docetaxel-loaded nanoparticles for the treatment of prostate cancer, Pharma 16 (1 ) (2024) 114, 10.3390/pharmaceutics16010114.
[17] Maresca KP , Hillier SM , Femia FJ , Keith D , Barone C , Joyal JL , Zimmerman CN , Kozikowski AP , Barrett JA , Eckelman WC , Babich JW , A series of halogenated heterodimeric inhibitors of prostate specific membrane antigen (PSMA) as radiolabeled probes for targeting prostate cancer, J. Med. Chem 52 (2 ) (2009) 347–357, 10.1021/jm800994j.19111054
[18] Gao H , Yang Z , Zhang S , Cao S , Shen S , Pang Z , Jiang X , Ligand modified nanoparticles increases cell uptake, alters endocytosis and elevates glioma distribution and internalization, Sci. Rep 3 (1 ) (2013) 2534, 10.1038/srep02534.23982586
[19] Wang J , Tian S , Petros RA , Napier ME , DeSimone JM , The complex role of multivalency in nanoparticles targeting the transferrin receptor for cancer therapies, J. Am. Chem. Soc 132 (32 ) (2010) 11306–11313, 10.1021/ja1043177.20698697
[20] Hong S , Leroueil PR , Majoros IJ , Orr BG , Baker JR , Holl MMB , The binding avidity of a nanoparticle-based multivalent targeted drug delivery platform, Chem. Boil 14 (1 ) (2007) 107–115, 10.1016/j.chembiol.2006.11.015.
[21] Susan L , Cheng G , 3, 3′-Diindolylmethane enhances apoptosis in docetaxel-treated breast cancer cells by generation of reactive oxygen species, Pharm. Biol 56 (2018) 407–414, 10.1080/13880209.2018.1495747.30301388
[22] Vaglienti MV , Subirada PV , Barcelona PF , Bonacci G , Sanchez MC , Quantification of reactive oxygen species using 2′7′-dichlorofluorescein diacetate probe and flow-cytometry in Müller glial cells, JoVE J. Vis. Exp 183 (2022) e63337.
[23] Mohammadpour R , Yazdimamaghani M , Cheney DL , Jedrzkiewicz J , Ghandehari H , Subchronic toxicity of silica nanoparticles as a function of size and porosity, J. Contr. Rel 304 (2019) 216–232, 10.1016/j.jconrel.2019.04.041.
[24] Muscella A , Vetrugno C , Migoni D , Biagioni F , Fanizzi FP , Fornai F , Marsigliante S , Antitumor activity of [Pt (O, O’-acac)(γ-acac)(DMS)] in mouse xenograft model of breast cancer, Cell Death Dis. 5 (1 ) (2014) e1014–e1014.24457958
[25] Wang Yan , Zeng San , Lin Tien-Min , Lisa Krugner-Higby Doug Lyman , Steffen Dana , Xiong May P. , Evaluating the anticancer properties of liposomal copper in a nude xenograft mouse model of human prostate cancer: formulation, in vitro, in vivo, histology and tissue distribution studies, Pharm. Res 31 (2014) 3106–3119, 10.1007/s11095-014-1403-6.24848339
[26] Tsukihara Hiroshi , Nakagawa Fumio , Sakamoto Kazuki , Ishida Keiji , Tanaka Nozomu , Okabe Hiroyuki , Uchida Junji , Matsuo Kenichi , Takechi Teiji , Efficacy of combination chemotherapy using a novel oral chemotherapeutic agent, TAS-102, together with bevacizumab, cetuximab, or panitumumab on human colorectal cancer xenografts, Oncol. Rep 33 (2015) 2135–2142, 10.3892/or.2015.3876.25812794
[27] Boinapally Srikanth , Ahn Hye-Hyun , Cheng Bei , Brummet Mary , Nam Hwanhee , Gabrielson Kathleen L. , Banerjee Sangeeta R. , Il Minn , Pomper Martin G. , A prostate-specific membrane antigen (PSMA)-targeted prodrug with a favorable in vivo toxicity profile, Sci. Rep 11 (2021) 7114, 10.1038/s41598-021-86551-1.33782486
[28] Watanabe R , Maekawa M , Kiyoi T , Kurata M , Miura N , Kikugawa T , Higashiyama S , Saika T , PSMA-positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state, Prostate 81 (16 ) (2021) 1390–1401, 10.1002/pros.24237.34516672
[29] Nakamura H , Takada K , Reactive oxygen species in cancer: current findings and future directions, Cancer Sci. 112 (10 ) (2021) 3945–3952, 10.1111/cas.15068.34286881
[30] Kosaka T , Hongo H , Miyazaki Y , Nishimoto K , Miyajima A , Oya M , Reactive oxygen species induction by cabazitaxel through inhibiting Sestrin-3 in castration resistant prostate cancer, Oncotarget 8 (50 ) (2017) 87675, 10.18632/oncotarget.21147.29152111
[31] Zou J , Xu MX , Li F , Wang YH , Li XQ , Yu DJ , Ma YJ , Zhang YY , Sun XD , Icaritin alleviates docetaxel-induced skin injury by suppressing reactive oxygen species via estrogen receptors, Thorac. Cancer 13 (2 ) (2022) 190–201, 10.1111/1759-7714.14245.34825483
[32] Zhu S , Liu S , Wang L , Ding W , Sha J , Qian H , Lu Y , Brusatol protects hepg2 cells against oxygen-glucose deprivation-induced injury via inhibiting mitochondrial reactive oxygen species-induced oxidative stress, Pharmacol 105 (7-8 ) (2020) 416–423, 10.1159/000504482.
[33] Xie J , Lai Z , Zheng X , Liao H , Xian Y , Li Q , Wu J , Ip S , Xie Y , Chen J , Su Z , Apoptotic activities of brusatol in human non-small cell lung cancer cells: involvement of ROS-mediated mitochondrial-dependent pathway and inhibition of Nrf2-mediated antioxidant response, Toxicol 451 (2021) 152680, 10.1016/j.tox.2021.152680.
[34] Hennrich U , Eder M , [177Lu] Lu-PSMA-617 (PluvictoTM): the first FDA-approved radiotherapeutical for treatment of prostate cancer, Pharmaceuticals 15 (10 ) (2022) 1292.36297404
[35] Fallah J , Agrawal S , Gittleman H , Fiero MH , Subramaniam S , John C , Chen W , Ricks TK , Niu G , Fotenos A , Wang M , FDA approval summary: lutetium Lu 177 vipivotide tetraxetan for patients with metastatic castration-resistant prostate cancer, Clin. Cancer Res 29 (9 ) (2023) 1651–1657, 10.1158/1078-0432.CCR-22-2875.36469000
[36] Wang X , Shirke A , Walker E , Sun R , Ramamurthy G , Wang J , Shan L , Mangadlao J , Dong Z , Li J , Wang Z , Schluchter M , Luo D , Wang Y , Stauffer S , Brady-Kalnay S , Hoimes C , Lee Z , Basilion JP , Small molecule-based prodrug targeting prostate specific membrane antigen for the treatment of prostate cancer, Cancers 13 (3 ) (2021) 417, 10.3390/cancers13030417.33499427
[37] Meher N , VanBrocklin HF , Wilson DM , Flavell RR , PSMA-targeted nanotheranostics for imaging and radiotherapy of prostate cancer, Pharma 16 (2023) 315, 10.3390/ph16020315.
[38] Gu F , Zhang L , Teply BA , Mann N , Wang A , Radovic-Moreno AF , Langer R , Farokhzad OC , Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers, Proc. Natl. Acad. Sci 105 (7 ) (2008) 2586–2591, 10.1073/pnas.0711714105.18272481
[39] Santino Federica , Stavole Pasquale , He Tingting , Pieraccini Silvia , Paolillo Mayra , Prodi Luca , Rampazzo Enrico , Gentilucci Luca , Preparation of non-toxic fluorescent peptide-coated silica/PEG nanoparticles from peptide-block copolymer conjugates, Micro 2 (2 ) (2022) 240–256, 10.3390/micro2020016.
[40] Woythe L , Madhikar P , Feiner-Gracia N , Storm C , Albertazzi L , A single-molecule view at nanoparticle targeting selectivity: correlating ligand functionality and cell receptor density, ACS Nano 16 (3 ) (2022) 3785–3796, 10.1021/acsnano.1c08277.35274534
[41] Elias DR , Poloukhtine A , Popik V , Tsourkas A , Effect of ligand density, receptor density, and nanoparticle size on cell targeting, Nanomed. Nanotechnol. Biol. Med 9 (2 ) (2013) 194–201, 10.1016/j.nano.2012.05.015.
[42] Perillo B , Di Donato M , Pezone A , , ROS in cancer therapy: the bright side of the moon, Exp. Mol. Med 52 (2020) 192–203, 10.1038/s12276-020-0384-2.32060354
[43] Yang H , Villani RM , Wang H , , The role of cellular reactive oxygen species in cancer chemotherapy, J. Exp. Clin. Cancer Res 37 (2018) 266, 10.1186/s13046-018-0909-x.30382874
[44] Adesina SK , Holly A , Kramer-Marek G , Capala J , Akala EO , Polylactide-based paclitaxel-loaded nanoparticles fabricated by dispersion polymerization: characterization, evaluation in cancer cell lines, and preliminary biodistribution studies, J. Pharm. Sci 103 (8 ) (2014) 2546–2555, 10.1002/jps.24061.24961596
[45] Tomayko MM , Reynolds CP , Determination of subcutaneous tumor size in athymic (nude) mice, Cancer Chemother. Pharm 24 (3 ) (1989) 148–154, 10.1007/BF00300234.
[46] Jensen MM , Jørgensen JT , Binderup T , , Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and reproducible than determined by 18F-FDG-microPET or external caliper, BMC Med. Imag 8 (2008) 16, 10.1186/1471-2342-8-16.
[47] Heuer Timothy S. , Ventura Richard , Mordec Kasia , Lai Julie , Fridlib Marina , Buckley Douglas , Kemble George , FASN inhibition and taxane treatment combine to enhance anti-tumor efficacy in diverse xenograft tumor models through disruption of tubulin palmitoylation and microtubule organization and FASN inhibition-mediated effects on oncogenic signaling and gene expression, EBioMedicine 16 (2017) 51–62, 10.1016/j.ebiom.2016.12.012.28159572
[48] Zhao XY , Yang S , Chen YR , Li PC , Dou MM , Zhang J , Resveratrol and arsenic trioxide act synergistically to kill tumor cells in vitro and in vivo, PloS One 9 (6 ) (2014) e98925, 10.1371/journal.pone.0098925.24901647
