A Novel PEtOx‐Based Nanogel Targeting Prostate Cancer Cells for Drug Delivery

Abstract

This study focuses on creating a specialized nanogel for targeted drug delivery in cancer treatment, specifically targeting prostate cancer. This nanogel (referred to as SGK 636/Peptide 563/PEtOx nanogel) is created using hydrophilic poly(2‐ethyl‐2‐oxazoline) (PEtOx) through a combination of living/cationic ring‐opening polymerization (CROP) and alkyne‐azide cycloaddition (CuAAC) “click” chemical reactions. A fluorescent probe (BODIPY) is also conjugated with the nanogel to monitor drug delivery. The characterizations through ¹H‐NMR, and FT‐IR, SEM, TEM, and DLS confirm the successful production of uniform, and spherical nanogels with controllable sizes (100 to 296 nm) and stability in physiological conditions. The biocompatibility of nanogels is evaluated using MTT cytotoxicity assays, revealing dose‐dependent cytotoxicity. Drug‐loaded nanogels exhibited significantly higher cytotoxicity against cancer cells in vitro compared to drug‐free nanogels. Targeting efficiency is examined using both peptide‐conjugated and peptide‐free nanogels, with the intracellular uptake of peptide 563‐conjugated nanogels by tumor cells being 60‐fold higher than that of nanogels without the peptide. The findings suggest that the prepared nanogel holds great potential for various drug delivery applications due to its ease of synthesis, tunable functionality, non‐toxicity, and enhanced intracellular uptake in the tumor region.

This study emphisizes an innovative PEtOx‐based nanogel tailored for targeted drug delivery in prostate cancer. Developed using click chemistry, a valuable technique for chemical synthesis, it exhibits consistent nanogel formation with customizable sizes. In vitro, drug‐loaded nanogel exhibits potent cytotoxicity against cancer cells. Notably, peptide‐conjugated nanogels significantly boost tumor cell uptake, showcasing promising promising potential for effective cancer drug delivey.

1. Introduction

Cancer is one of the most important health problems of today because it is common and fatal. Although significant advances have been made in the field of cancer treatment in recent years, traditional cancer treatment methods are still insufficient to effectively target cancer cells and avoid damaging healthy tissues.^{[ 1 ]} Recently, a wide variety of nanoplatforms have been developed that show good biocompatibility, high drug loading capacity, controlled drug release ability, as well as enhanced tumor penetration ability.^{[ 2 ]} To address this challenge, targeted drug delivery approaches offer a promising solution. Nanotechnology represents a significant advance in this sense. Nano‐sized gel particles, known as nanogels, are utilized to enable precise drug delivery. Nanogels are promising materials due to their advantageous characteristics. They are easily synthesized and possess high stability, controlled size, efficient drug encapsulation, and prolonged drug release capabilities. Their small size contributes to their permeation ability. Nanogels can demonstrate both active and passive drug targeting, low toxicity, biomolecule identification, and response to various environmental factors like pH, temperature, light, magnetic field, and ionic strength. Additionally, their viscoelasticity, biocompatibility, and biodegradability further enhance their appeal for various applications.^{[ 3 ]}

Effective cancer treatments require effective surface modification and multifunctional chemical modification‐mediated nanomaterials.^{[ 4 ]} This is a crucial step in increasing targeting ability and minimizing non‐specific interactions with healthy tissues. Numerous studies have shown that peptides containing specific amino acid sequences associated with cancer cells can be effectively used to selectively target cancer cells when combined with various drug delivery systems. By binding relevant cancer‐specific peptides to nanogels, the drug can be directly delivered to the desired site.^{[ 5} , ⁶ , ⁷ , ⁸ , ^{9 ]}

To create an effective nanogel theranostic platform, this system should be functional under mild conditions.^{[ 10 ]} Tumor cells exhibit a widespread overexpression of glutathione (GSH), resulting in significantly heightened cellular redox sensitivity in these cells compared to normal ones. This particular characteristic of being responsive to elevated GSH levels significantly enhances the attractiveness of the disulfide‐crosslinking strategy for both cancer treatment and diagnosis. Disulfide bonds can be effectively cleaved when exposed to high GSH concentrations in tumor cells, enabling controlled drug release.^{[ 11 ]} In this context, a novel nanogel system could be a significant step forward. However, it's important to note that only a limited number of therapeutic nano‐carrier systems have received Food and Drug Administration (FDA) approval for clinical use.^{[ 12 ]} Currently, polymeric nano‐carrier systems are undergoing rigorous testing in numerous clinical trials.^{[ 13 ]}

Polymeric nanoparticles offer a promising avenue for therapeutic applications due to their potential benefits, including an extended drug lifecycle, reduced toxicity, fewer side effects, improved biodistribution, and enhanced solubility.^{[ 14 ]} Numerous studies have explored various synthesis strategies for producing nanogels using different types of polymeric materials.^{[ 15 ]} Most drug carrier systems are designed as polyethylene glycol (PEG)‐conjugated nanocarriers to prevent non‐specific interactions between drugs and components in healthy tissues and the bloodstream.^{[ 16} , ¹⁷ , ^{18 ]} However, it is important to note that PEG is not without its drawbacks as a material for drug delivery systems. It can lead to unwanted side effects such as the formation of antibodies against PEG (anti‐PEG), hypersensitivity reactions, and vacuolation.^{[ 19} , ²⁰ , ^{21 ]} These reported side effects have raised safety concerns regarding the use of PEG as a polymer. Polyoxazolines (POx)s offer an alternative to PEG due to their hydrophilicity, antifouling properties,^{[ 21} , ^{22 ]} and biocompatibility ^{[ 23} , ²⁴ , ^{25 ]} A significant advantage of (POx)s over PEG lies in their versatile functionalization chemistry, which enables the creation of a wide range of functional polymers and materials.^{[ 26 ]} To date, POx‐based nanoparticles have not been widely utilized as a cancer drug delivery system. However, it's worth noting that clinical trials for the first drug delivery system based on POx‐derived nanoparticles are currently underway (SER‐214, phase I).^{[ 27 ]}

In this study, we report the production and functionalization of multifunctional Poly(2‐ethyl‐2‐oxazoline) (PEtOx)‐based nanogels. These nanogels have been conjugated with a targeting group, PSMA peptide 563, and a near‐infrared fluorescence (NIRF) group (BODIPY). As a result, the nanogel has been turned into a polymer with fluorescent properties and loaded with the antitumor drug SGK 636. A previous study reported the synthesis of an active pharmaceutical ingredient called SGK 636, whose synthesis and in vivo activity against prostate cancer were determined. SGK 636 is an original molecule synthesized by Birgul and colleagues, containing thioether‐triazole and S‐Naproxen structures, and its in vivo efficacy against prostate cancer has also been established.^{[ 28 ]} This compound has been designed to inhibit the Methionine Aminopeptidase‐2 (MetAP2) enzyme in prostate cancer. The prepared nanogel was characterized using dynamic light scattering (DLS) and environmental scanning electron microscopy (ESEM). Furthermore, the drug content and efficacy of these nanogels in prostate cancer cells were investigated. In vitro cell viability tests and cellular uptake studies were conducted at various doses in normal prostate epithelial cells (PNT1A) and human prostate carcinoma cells (LNCaP and DU‐145).

2. Experimental Section

2.1. Materials

All Fmoc‐protected l‐amino acids, coupling reagents, and Fmoc‐Ser(Trt)‐Wang resin (100–200 mesh, 0.3–0.8 meq g⁻¹ loading capacity) were obtained from Chem‐Impex (USA). 1,1,1‐tris(4‐hydroxyphenyl) ethane (99%, Sigma‐Aldrich), tetrahydrofuran (THF, containing 250 ppm BHT as an inhibitor, puriss. P.a., ACS reagent, reagent Ph. Eur., ≥ 99%, Sigma‐Aldrich), Triethylamine (TEA,(Et₃N, Merck Chem.), 6‐bromohexanoyl chloride (97%, Sigma‐Aldrich), sodium hydrogen carbonate (NaHCO₃, Tekkim), Na₂SO₄ (99.0–100.5%, Sigma‐Aldrich), 2‐ethyl‐2‐oxazoline (EtOx, 99%, Alpha Aesar), sodium azide (NaN₃, 99.5%, Sigma‐Aldrich), copper(I) bromide (Cu(I)Br, 99.9% trace metals basis, ALDRICH), N,N‐dimethylformamide (DMF, reagent grade, VWR Chem.), acetonitrile (CH₃CN, ≥99.8%, 8149), pentamethyldiethylenetriamine (PMDETA, >99.0% (GC), TCI), cystamine dihydrochloride (96%, ALDRICH), potassium hydroxide (KOH, 85–100.5%, Sigma‐Aldrich), methanol (CH₃OH, ≥ 99.7%, Sigma‐Aldrich), propargyl bromide (80% solution by weight in toluene, stabilized, ACROS Organics), copper sulfate (CuSO₄, 99%, Sigma‐Aldrich), l‐ascorbic acid sodium salt (USP grade, Amresco), dialysis membrane (MWCO 3500, Spectra/Por), toluene, DCM, ACN, and EtOx were dried over CaH₂ overnight and then distilled under vacuum. They were subsequently stored with molecular sieves dried at high temperatures under N₂. All other chemicals were reagent‐grade and used without further purification unless otherwise stated.

2.1.1. Cell Lines

The PNT1A human prostate epithelial cells were sourced from Sigma Aldrich, USA. Human prostate carcinoma cells (DU‐145 and LNCaP) were procured from the American Type Culture Collection (ATCC). All cell lines were cultured and maintained in Roswell Park Memorial Institute 1640 Medium (RPMI 1640, Gibco), containing 10% (v/v) fetal bovine serum (FBS; Gibco) and 100 units/mL penicillin/streptomycin (Gibco). Cell cultures were maintained in a humidified incubator set at 37 °C with 5% CO₂.

2.2. Instruments and Methods

Peptide 563 was synthesized using the Fmoc‐based solid‐phase peptide synthesis (SPPS) strategy on a Rink amide resin with the CEM Discover Bio‐Manual Microwave Peptide Synthesizer.

Purification of the peptide was carried out via reverse phase preparative HPLC, utilizing a Dionex UltiMate 3000 HPLC system equipped with a Thermo Scientific Hypersil Gold C18 column (250 × 10 mm, 5 µm) at a flow rate of 2 mL min⁻¹ at 40 °C. The eluent consisted of acetonitrile/water with a gradient containing 0.1% trifluoroacetic acid (5–100%, 1–70 min). The peptide was initially dissolved in MilliQ water containing 10% acetic acid, and the peptide concentration was adjusted to 20 mg mL⁻¹. Purification was performed with a 2 mL injection loop, and this process was repeated consecutively. The purity of each fraction was assessed using analytical RP‐HPLC before combining the pure fractions. Analytical HPLC was conducted on a Dionex UltiMate 3000 HPLC system equipped with a Thermo Scientific AcclaimTM 120 C18 column (46 × 150 mm, 3 µm), using an acetonitrile/water gradient containing 0.1% trifluoroacetic acid (5–100%, 1–30 min) with a flow rate of 0.4 mL min⁻¹. After confirming the purity of the collected fractions through analytical HPLC, the peptide was lyophilized using a Telstar Cryodos Freeze Dryer and stored at −20 °C.

Mass analysis was performed using mass spectrometry on an Agilent 6530 Q‐TOF mass spectrometer equipped with an electrospray ionization (ESI) source. The concentration of synthesized peptide was adjusted to 1 mg mL⁻¹ with water for characterization.

Proton nuclear magnetic resonance (¹H NMR) spectra were recorded at 298.15 K in (CD₃)₂SO or CDCl₃ using a Varian 600 Spectrometer operating at 600 MHz. Chemical shifts were reported in ppm. ¹H NMR was employed to confirm the structure of the products and detect changes in chemical shifts.

FT‐IR measurements were conducted on a Perkin–Elmer FTIR System Spectra BX in the range of 4500–450 cm⁻¹ in transmittance mode. This analysis showed changes in functional groups and peak intensities in the spectra.

Gel permeation chromatography (GPC) was employed to determine the average molecular weight of the PEtOx polymer using an Agilent instrument (Model 1100) with a Viscotek TDA302 GPC system, a Viscotek GPCmax pump, a refractive index detector, and a Tosoh TSKGel G3000PWxl column (300 mm × 7.8 mm) at 25 °C. The mobile phase consisted of PBS (pH = 7.0) with 0.05% NaN₃, and the flow rate was 1 mL min⁻¹. The system was calibrated using a PEO standard solution (M _p = 135 000 g mol⁻¹, M _w/M _n = 1.05) obtained from Viscotek. All samples were filtered using 0.45 µm regenerated nylon cellulose syringe filters.

Particle size and size distribution of nanogel formulations were determined using the dynamic light scattering (DLS) technique, while zeta potentials were measured based on electrophoretic mobility. Each sample measurement was repeated three times.

HPLC, equipped with a photodiode array (PDA) detector at 332 nm, was used for encapsulation experiments. Separation was carried out on a C‐18 column (4.6 × 250 mm) with a 5 µm particle diameter, and the mobile phase was a mixture of 85% acetonitrile and 15% water at a flow rate of 1.0 mL min⁻¹. The main stock solution of SGK 636 was prepared in methanol at a concentration of 1000 mg L⁻¹, with all dilutions made using methanol.

For the morphological examination of the nanogels, an FEI brand QUANTA FEG 250 ESEM model environmental scanning electron microscope was used. Images were captured at 30 kV voltage and 3.5 spot values with an EDT detector (Everhart‐Thornley detector, high vacuum detector). Nanogels were deposited on an aluminum foil placed on carbon tape with a few drops of suspension diluted with water and dried under vacuum conditions at room temperature. The dried nanogels were coated with platinum vapor before examination in SEM.

For the morphological examination of the nanogels, transmission electron microscopy (TEM) (JEOL JEM 2100 microscope) was also used. Images were recorded at an accelerating voltage of 200 kV. TEM samples were prepared by applying a drop of nanogel solution onto a carbon‐coated copper TEM grid (200 mesh size). The samples were dried at room temperature for 1 h before imaging.

Fluorescence spectra were obtained by diluting the nanogels with distilled water using the FS5 spectrofluorometer (Edinburgh Instruments, Livingston, UK).

2.2.1. Statistical Analysis

All data were presented as the mean ± standard deviation (SD) of the mean. Statistical analysis was performed using a t‐test and p values <0.05 were considered statistically significant.

2.3. Synthesis

2.3.1. Synthesis of Ethane‐1,1,1‐Triyltris (Benzene‐4,1‐Diyl) Tris (6‐Bromohexanoate)

1,1,1‐Tris (4‐hydroxyphenyl) ethane (3.2 mmol), dry THF (115 mL), and TEA (1.5 mL) were added to the reaction flask and stoppered. Then, by placing it in a water‐ice bath, the temperature of the mixture was reduced by 0–5 °C, while vacuum‐nitrogen treatment was performed three times to provide an inert atmosphere. 6‐Bromohexanoyl chloride (10.6 mmol) was added dropwise under nitrogen. After the addition was complete, the reaction continued at room temperature for 14 h. It was then placed in an oil bath set at 60 °C. At the end of the reaction, the solvent was evaporated using a rotary evaporator. It was dissolved in dichloromethane, and extracted with NaHCO₃ (aq) and purified with water, respectively. The organic phases were collected and dried over Na₂SO₄ (s) and then removed by simple filtration, and the solvent was evaporated using a rotary evaporator. It was dried in a vacuum oven at 40 °C for 24 h. Ethane‐1,1,1‐triyltris (benzene‐4,1‐diyl)tris (6‐bromohexanoate) was characterized by ¹H‐NMR and FT‐IR spectra. ¹H‐NMR (CDCl₃, 600 MHz, δ, ppm): 1.83–1.92 (18H, m, ─CH₂─CH₂─CH₂─), 2.14 (3H, s, CH₃─C─), 2.58–2.55 (6H, t, ─OCO─CH₂─CH₂─), 3.55 (6H, t, Br─CH₂─CH₂─), 7.08–6.96 (12H, m, Ar). FT‐IR, v _max (cm⁻¹): 2939 (─CH₂ stretching); 1783 (─OC═O); 1605, 1504 (aromatic C═C); 1203–1114 (C─O); 550 (─C─Br) cm⁻¹ (see Figure S3, Supporting Information).

2.3.2. Synthesis of α‐Azide Triarm‐Terminated Star‐Shaped Poly (2‐Ethyl‐2‐Oxazoline) [Star‐(PEtOx‐N₃)₃]

The moisture in the reaction tube was dried using a heat gun, closed with a silicone stopper, and degassed with dry nitrogen. Ethane‐1,1,1‐triylthris (benzene‐4,1‐diyl) tris (6‐bromohexanoate) (0.41 mmol) initiator and acetonitrile (15 mL) were added to the flask, respectively. 2‐Ethyl‐2‐oxazoline (5 mL, 49.5 mmol) monomer was introduced into the stirring solution, and the reaction was continued at 130 °C for 16 h through living cationic ring‐opening polymerization. The reaction mixture was then cooled to room temperature, and the living polymer chains were terminated by adding sodium azide (0.4 g, NaN₃) and stirred for 72 h at 65 °C. After termination, the mixture was passed through a silica gel column using DCM solvent to remove excess NaN₃. After removing the solvent under reduced pressure using a rotary evaporator, the polymer was dissolved in a minimum amount of DCM and precipitated in cold diethyl ether. The resulting polymer was then dried in a vacuum oven for 1 day. M _n(GPC) = 9248, M _w(GPC) = 10 314, M _w/M _n = 1.11, f _(GPC) = 3.02. ¹H‐NMR (DMSO‐d₆,600 MHz, δ, ppm): 0.93 (3H, ─NCOCH₂CH₃), 1.49 (2H, OCO─CH₂─CH₂─CH₂), 1.62 (3H, CH₃─C), 2.29–2.24 (2H,─NCOCH₂─), 3.40 (4H, ─NCH₂CH₂N─), 7.03–6.59 (12H, aromatic protons). FT‐IR, v _max (cm⁻¹): 2977–2878 (C─H, stretching); 2097 (─N₃); 1752 (─OC═O); 1632 (─NC═O); 1540, 1503 (aromatic, C═C); 1464 (─CH₂, bending); 1375 (─CH₃, bending); 1237–1171 (C─N, stretching) cm⁻¹ (see Figures S4 and S5, Supporting Information).

2.3.3. Synthesis of BODIPY Derivative Dye (BODIPY Compound‐Alkynyl)

2,4‐Dimethylpyrrole (0.63 g, 6.6 mmol) and 4‐hydroxybenzaldehyde (0.37 g, 3.0 mmol) were dissolved in 90 mL of THF. A few drops of trifluoroacetic acid (TFA) were added, and the mixture was stirred at room temperature for 12 h. Subsequently, 2,3‐dichloro‐5,6‐dicyano‐p‐benzoquinone (DDQ) (0.68 g, 3.0 mmol) dissolved in 120 mL of THF was added dropwise to the reaction mixture and stirred for 4 h. Triethylamine (18 mL, 0.13 mol) was added to the mixture, followed by the dropwise addition of BF₃.OEt₂ (18 mL, 0.15 mol) to the reaction medium in an ice bath. The mixture was then stirred at room temperature for 12 h. The solvent was removed by applying a vacuum to the rotary evaporator. The reaction mixture was purified on a silica gel‐filled column using dichloromethane as the mobile phase. 200 mg (0.58 mmol) 4,4′‐difluoro‐8‐(4‐hydroxy)‐phenyl‐1,3,5,7‐tetramethyl‐4‐bora‐3a, 4a‐diaza‐s‐indacene and 260 mg (1.46 mmol) 4‐diethylaminobenzaldehyde was dissolved in toluene in a reaction flask. A catalytic amount of Mg(ClO₄)₂, piperidine (1 mL) and glacial acetic acid (1 mL) at a 1:1 ratio were added into the reaction mixture and the mixture was boiled for 4 h in a Dean–Stark trap system. At the end of this period, toluene was distilled off, and the resulting substance was purified using a column filled with silica gel in a 5:1 hexane/ethyl acetate solvent system. 60 mg (0.091 mmol) of 4,4′‐difluoro‐8‐(4‐hydroxy) phenyl‐1,7‐dimethyl‐3,5‐ (4‐4ʺ diethyl aminophenyl) ethenyl)−4‐bora‐3a, a mixture of 4a‐diaza‐s‐indacene and 25 mg (0.182 mmol) of K₂CO₃ were placed in a Schlenk tube in DMF. Then, 0.012 mL (0.1 mmol) of 3‐bromo‐1‐propine was added. This mixture was stirred under an argon atmosphere at room temperature for 24 h. At the end of this period, the mixture was extracted with dichloromethane/water to remove DMF. The crude product obtained was purified by chromatography using CH₂Cl₂. The analysis results for the BODIPY‐derived compound were obtained in accordance with the values given in the literature.^{[ 29 ]}

2.3.4. Conjugation of BODIPY Comp‐Alkynyl to α‐Azide Triarm Star‐Shaped Poly (2‐Ethyl‐2‐Oxazoline) [Star‐(PEtOx‐N₃)₃]

For the conjugation, click coupling reactions of star‐(PEtOx‐N₃)₃ and BODIPY‐alkynyl were conducted using a CuBr/PMDETA catalyst. 7 mg (0.05 mmol) of Cu(I)Br was added to a Schlenk tube, which was then sealed, degassed, and filled with nitrogen. In a separate vial, 102 mg (0.017 mmol) of star PEtOx polymer, 10 mg (0.017 mmol) of BODIPY, and 43 µL (0.20 mmol) of pentamethyldiethylenetriamine (PMDETA) were dissolved in 15 mL DMF and filled with nitrogen. The mixture was then transferred to the Schlenk tube, and nitrogen was introduced after a vacuum‐nitrogen cycle. The click reaction was carried out at room temperature for 24 h on the magnetic stirrer. After the reaction was completed, it was purified using a dialysis membrane with a 2000 Da cut‐off against a methanol/water mixture for 24 h. BODIPY‐conjugated polymer solution was then dried using the freeze‐dryer. ¹H‐NMR (DMSO‐d₆, 600 MHz, δ, ppm): 7.87 (1H, Triazole‐H), 7.47–6.61 (Ar─H), 4.59–4.51 (1H, CH═CHCH₃), 3.96–3.94 (2H, ─NCH₂CH₃), 3.40 (4H, ─NCH₂CH₂N─), 2.29–2.24 (2H, ─NCOCH₂─), 1.62 (3H, ─CCH₃), 1.50 (2H, OCOCH₂CH ₂ ─), 1.21 (3H, ─NCH₂CH₃), 0.92 (3H, ─NCOCH₂CH ₃ ). FT‐IR, v _max (cm⁻¹): 2978–2840 (C─H, stretching); 2100 (─N₃); 1728 (─OC═O); 1621 (─NC═O); 1522 (B─N); 1471–1422 (─CH₂, bending); 1375 (─CH₃, bending); 1197 (C─N, stretching) cm⁻¹.

2.3.5. Synthesis of N, N'‐(Disulfandiylbis (Ethane‐2,1‐Diyl)) bis (Prop‐2‐yn‐1‐Amine) (BAC) Crosslinker

Cystamine dihydrochloride (22.2 mmol) was dissolved in 50 mL of water. Then, 0.9 mol of KOH was added and mixed. After that, 10 mL of TEA and CH₃OH (50 mL) were added. The reaction flask was sealed with a stopper, and it was subjected to vacuum nitrogen three times before being stirred at room temperature for 2 h. Propargyl bromide (19.8 mmol) was added drop by drop. Once the addition was complete, the reaction mixture was placed in an oil bath set at 80 °C, and the reaction continued for 48 h. After the reaction flask had cooled, the triethylamine salts were removed by filtration, and the solvent was evaporated using rotary evaporation. The resulting material was then dissolved in dichloromethane and extracted successively with aqueous NaHCO₃ and purified with water, respectively. The organic phases were collected and dried using Na₂SO₄. The Na₂SO₄ was removed by filtration, and the solvent was evaporated on the rotary evaporator. Finally, the product was dried in a vacuum oven at 40 °C. ¹H‐NMR (CDCl₃, 600 MHz, δ, ppm): 3.47 (4H, s, ─NHCH₂C≡CH), 3.03 (4H, t, ─SCH₂CH₂NH─), 2.88 (4H, t, ─SCH₂), 2.25 (2H, s, CH≡C─) ppm. FT‐IR, v _max (cm⁻¹): 3287 (N─H, stretching); 2919–2816 (C─H stretching); 2103 (C≡CH, stretching); 1647–1607 (N─H, bending); 1440 (─CH₂, bending); 1328–1280 (C─N, stretching) cm⁻¹.

Synthesis of Active Targeting Agent‐PSMA‐563 Peptide

The PSMA‐563 coded peptide was synthesized using Wang resin loaded with 0.2 mmol Fmoc‐Ser (tBu)‐OH through a Fmoc(fulorenylmethyloxycarbonyl) based solid‐phase peptide synthesis (SPPS) strategy (Figure  1 ). This synthesis was carried out using a CEM Discover Bio‐Manual Microwave Peptide Synthesizer.^{[ 44 ]}

Figure 1.

Synthesis of PSMA‐563 peptide (alkyne‐GRFLTGGTGRLLRIS).

4‐Pentynoic acid was added to the N‐terminus of the peptide to enable its attachment to the nanogel through a Cu (I) catalyzed azide‐alkyne cycloaddition (CuAAC) reaction. All arginines were incorporated into the peptide chain using the stronger coupling reagent HCTU [2‐(6‐chloro‐1‐H‐benzotriazol‐1‐yl)−1,3,3,6‐tetramethyl aminiumhexafluorophosfat]. Other amino acids and 4‐pentynoic acid were conjugated with HBTU.

2.3.6. Preparation of the Nanogels

Preparation of Bodipy Conjugated Nanogel Formulations Containing the Active Ingredient (SGK 636), NG4 Nanogel

4.6 mg of Cu(I)Br was added to the reaction flask. For nanogels containing the active substance, 12 mg of SGK 636 was included. Then, a mixture of 60 mg of star‐shaped PEtOx polymer, 40 mg of BODIPY‐conjugated star PEtOx polymer and 5.5 mg of cross‐linker [N,N'‐(disulfandiylbis(ethane‐2,1‐diyl))bis(prop‐2‐yn‐1‐amine)] were dissolved in 60 mL of dichloromethane (DCM), and 40 µL of pentamethylethylenetriamine (PMDETA) was added to the solution. The formulation was stirred for 1 h in a magnetic stirrer. Subsequently, sonication was applied for 1 min at 15‐s intervals. When the reaction was complete, DCM was evaporated using a rotary evaporator, and the resulting mixture was dispersed in 20 mL of distilled water to obtain nanogel formulations. The dispersed formulation was dialyzed (MWCO 3500) against distilled water. 10 mL was taken from the dialysis tube and dried using the lyophilization method. The remaining 10 mL of the dispersed reaction mixture was used for the synthesis of active ingredient‐containing peptide‐coupled nanogels. ¹H‐NMR (DMSO‐d₆, 600 MHZ, δ, ppm): 7.87 (1H, triazole H), 7.62–6.59 (Ar─H), 4.24–4.09 (1H, ─SCH₂─), 3.82 (3H, ─OCH₃), 3.40 (4H, ─NCH₂CH₂N─), 2.64 (2H, ─CH₂SS─), 2.30–2.25 (2H, ─NCOCH₂─), 2.05 (9H, Ar─CH₃), 1.64 (3H, ─CHCH₃), (3H, ─NCH₂CH₃), 1.21 (3H, ─NCH₂CH₃), 0.92 (3H, NCOCH₂CH₃). FT‐IR, v_max (cm⁻¹): 2978–2941 (C─H, stretching); 2100 (─N₃); 1728 (─OC═O); 1621 (─NC═O); 1522 (B─N); 1471–1422 (─CH₂, bending); 1375 (─CH₃, bending); 1290 (C─S); 1197 (C─N, stretching); 498, 442 (S─S) cm⁻¹.

Preparation of Bodipy and Peptide‐Conjugated Nanogel Formulations Containing the Active Ingredient (SGK 636), NG6 Nanogel

The remaining 10 mL of dispersed BODIPY‐conjugated nanogel containing active ingredient was sonicated for 1 min at 40% amplitude. After sonication, 2 mg of prostate cancer‐specific targeted peptide 563 was added, and the total volume was adjusted to 30 mL by adding distilled water. Then, 2.5 mg of copper sulfate (CuSO₄) and 10 mg of sodium ascorbate (NaAsc) were introduced, and the mixture was left to incubate for 2 days. After the incubation period, it was transferred to a dialysis membrane (MWCO 3500) and dialyzed with 0.1 M EDTA for 1 day, followed by dialysis against distilled water for an additional day. The sample was then freeze‐dried in a lyophilizer for 72 h. ¹H‐NMR (DMSO‐d₆, δ, ppm): 8.05 (1H, ─NH), 7.63‐6.59 (Ar─H), 3.82 (3H, ─OCH₃), 3.48 (2H, Ser (─CH₂OH)), 3.40 (4H, ─NCH₂CH₂N─), 2.64 (2H, ─CH₂SS─), 2.30–2.25, (2H, ─NCOCH₂─), 2.04 (9H, Ar─CH₃), 1.64 (3H, ─CHCH₃), 1.21 (3H, Thr‐CH₃; 3H, ─NCH₂CH₃), 1.50 (2H, OCOCH₂CH₂─), 1.21 (3H, ─NCH₂CH₃), 1.12 (3H, Iso‐CH₂), 0.93 (3H, ─NCOCH₂CH₃), 0.83 (3H, Iso, Leu‐CH₃) ppm. FT‐IR, v_max (cm⁻¹): 3520–3383 (─NH, ─OH); 3027–3004 (C─H stretching); 1736 (─OC═O); 1668 (NHC═O); 1614 (─NC═O); 1510 (B─N); 1474–1392 (─CH₂ bending); 1353 (─CH₃ bending); 1290 (C─S); 1193 (C─N stretching); 498, 442 (S─S) cm⁻¹.

Preparation of Blank (NG1 Nanogel) or SGK 636 Loaded Nanogel (NG2 Nanogel) Formulations with Star‐Shaped PEtOx Polymer

In the reaction flask, 2.3 mg of Cu(I)Br was introduced. For nanogels containing the active substance, 6 mg of SGK 636 was also added. Following this, 50 mg of star‐shaped PEtOx polymer and 2.8 mg of crosslinker were dissolved in 30 mL of dichloromethane (DCM), and 20 µL of pentamethylethylenetriamine (PMDETA) was incorporated into the solution. The formulation was stirred for 1 h on a magnetic stirrer. Subsequently, sonication was applied for 2 min. After the reaction was completed, DCM was removed under low pressure using a rotary evaporator. The resulting gel was then dispersed in 10 mL of distilled water to obtain nanogel formulations. This dispersed formulation was subjected to dialysis first against 0.1 M EDTA and then against distilled water. After the dialysis step, the nanogel formulation was freeze‐dried.

2.3.7. Physical Stability of SGK 636 Loaded Nanogel (NG2 Nanogel) Formulations in Different Media

After the evaporation step, the prepared nanogel formulations were re‐suspended in 20 mL of distilled water. This suspension was then subjected to centrifugation at 10 000 rpm for 2 h, and the supernatant was carefully decanted. The resulting nanogels were subsequently re‐suspended in three different media: distilled water, phosphate‐buffered saline with a pH of 7.4 (PBS), or a solution containing 1% fetal bovine serum (FBS). The particle size of the nanogel formulations was measured at specific time points (0, 0.5, 1, 2, and 3 h).

Determination of Encapsulation Efficiency of SGK 636 Loaded Nanogel (NG2) Formulations

To extract SGK 636, lyophilized nanogels were mixed with 10 mL of methanol using a magnetic stirrer for 6 h. Afterward, the resulting mixture was centrifuged for 2 h at 9500 rpm. The supernatant was carefully collected and passed through a 0.45 µm membrane filter. Subsequently, the filtered solution was analyzed at 332 nm (for SGK 636 content) using high‐performance liquid chromatography (HPLC). The encapsulation efficiency was calculated using the following equation (n = 3):

$$\begin{array}{ccc}& & \mathrm{Encapsulation}\mathrm{Efficiency}\left(\%\mathrm{EE}\right)\hfill \\ & & =\frac{\mathrm{Actual}\mathrm{amount}\mathrm{of}\mathrm{SGK}636\mathrm{in}\mathrm{the}\mathrm{nanogels}}{\mathrm{Theoretical}\mathrm{amount}\mathrm{of}\mathrm{SGK}636\mathrm{in}\mathrm{the}\mathrm{nanogels}}\times 100\hfill \end{array}$$ (1)

2.3.8. Cytotoxicity Activities of Empty Nanogel (NG1 Nanogel) and SGK 636‐Loaded Nanogel (NG2 Nanogel)

Initially, a WST‐1 assay to assess the impact of empty nanogel on PNT1A cell proliferation was conducted. To do this, cells were seeded in a 96‐well plate with a density of 5 × 10³ cells per well overnight and then exposed to varying concentrations of cargo‐free (empty) nanogel, specifically 150, 200, 300, 350, 400, 450, and 750 µg mL⁻¹. After 72 h, the WST‐1 cell proliferation assay was performed using the kit (Sigma Aldrich, #5 015 944 001), following the manufacturer's instructions. The absorbance values were measured at 450 nm with a microplate reader, using 630 nm as a reference wavelength, and calculated the percentage of cell viability by normalizing the results to untreated cells, which were considered 100%. Subsequently, the maximum possible carrier doses were determined and the proliferation assay was conducted using DU‐145 and LNCaP cells to evaluate the cytotoxic effect of SGK 636‐loaded nanogels. Given that 15 mg of PEtOx nanogel contains 0.937 mg of the SGK 636 molecule, the IC₅₀ values were adjusted as indicated in Table 2. LNCaP cells were exposed to concentrations of 10, 17, and 25 µg mL⁻¹, while DU‐145 cells were treated with concentrations ranging from 50 to 350 µg mL⁻¹ of SGK 636‐loaded nanogel. Following 24, 48, and 72 h of incubation, the WST‐1 cell proliferation assay was conducted and cell viability was calculated.

Table 2.

IC₅₀ values of free SGK 636 (NG1 nanogel) and SGK 636‐loaded PEtOx nanogel (NG2 nanogel) for DU‐145 and LNCaP prostate cancer cell lines.

	DU‐145	LNCaP
PEtOx Nanogel (SGK636 free) (NG1)	5.2801 µg mL−1	1.144 µg mL−1
SGK636 loaded‐PEtOx Nanogel (NG2)	81.53 µg mL−1	17.31 µg mL−1

2.4. Flow Cytometry

The targeting efficiency of the Peptide563 and BODIPY conjugated‐PEtOx Nanogel (SGK 636 free) (NG5 nanogel) was assessed using flow cytometry. To conduct this experiment, PNT1A normal prostate epithelial cells, along with LNCaP and DU‐145 prostate cancer cells, were seeded in a 6‐well plate at a density of 5 × 10⁵ cells per well and allowed to attach overnight. The following day, the cells underwent a 6h treatment with NG5 nanogel. After the treatment period, the cells were harvested by trypsinization and subsequently fixed using a 0.05% (v/v) formaldehyde solution. The targeting profile of NG5 nanogel was determined using the Becton Dickinson FACSCalibur flow cytometer and analyzed with the CellQuestPro software (Becton Dickinson, USA).

3. Results

3.1. Synthesis and Characterization of Polymer

Here, we present the synthesis of a novel PEtOx‐based nanogel designed for active targeting through the incorporation of fluorescein (a near‐infrared fluorescent (NIRF) dye) and peptide 563 (a targeting peptide overexpressed in prostate cancer cells) (Figure  2 ). The synthesis process commenced with the creation of an initiator, which was confirmed through prominent features in the FT‐IR and ¹H NMR spectra (Figure S3, Supporting Information). In the FT‐IR spectrum, distinctive bands at 1783 and 550 cm⁻¹ indicated the presence of ester (─OC═O) and C─Br groups, respectively. In the ¹H NMR spectrum, the initiator exhibited signals for three aromatic protons and methylene protons [phenyl‐(CH₂)₅‐Br]₃, appearing at ≈7.08–6.96 ppm and ≈3.55–1.83 ppm, respectively. A signal at 2.58 ppm confirmed the presence of the ester‐bound ─CH₂ group. Subsequently, a three‐armed PEtOx‐based star polymer was synthesized via a living cationic ring‐opening polymerization of 2‐ethyl‐2‐oxazoline, employing a three‐armed bromo‐functional initiator (etan‐1,1,1‐triyltris(benzene‐ 4,1‐did)tris(6‐bromohexanoate)). The terminal of the living PEtOx polymer was capped with sodium azide. The synthesis was characterized by GPC, FT‐IR, and ¹H NMR spectra. The GPC chromatogram indicated a number‐average molecular weight (M _n) value of 9248 g mol⁻¹, displaying monomodal traces and low polydispersity indexes (PDI = 1.11) (Figure S5a, Supporting Information). The functionality (number of polymer arms) was calculated as f _(GPC) = 3.02 (Figure S5b, Supporting Information). The ¹H NMR spectrum revealed the appearance of new proton peaks at ≈0.93, ≈2.24–2.29, and ≈3.40 ppm corresponding to the polymer side group and the polymer backbone. Proton signals at ≈7.03–6.59 ppm and ≈2.15–1.83 ppm indicated the initiator (as shown in Figure S3, Supporting Information). Further details of the FT‐IR spectrum for star‐shaped PEtOx are provided in Figure S4, Supporting Information. Specific wavenumbers in the FT‐IR spectra of PEtOx at 2977, 2878, 1464, and 1375 cm⁻¹ were assigned to CH₃ stretching, CH₂ symmetric stretching, CH₂ bending vibration, and CH₃ bending vibration, respectively. The presence of vibration at 2097 cm⁻¹ clearly indicates the end group ─N₃ of PEtOx. The strong band at 1632 cm⁻¹ corresponds to C═O stretching of the amide. The bands at 1752 cm⁻¹ and in the region 1540–1503 cm⁻¹ correspond to ─OC═O (ester group) and the stretching of the C═C aromatic double bond of the initiator, respectively. The PEtOx polymer was subsequently modified with fluorescent dye BODIPY ^{[ 30 ]} using azide‐alkyne CuAAC click chemistry to enable active targeting for imaging purposes. BODIPY was prepared with an alkyne functional group. The successful conjugation was confirmed through FT‐IR and ¹H NMR spectra. The azide peak intensity decreased at 2100 cm⁻¹, and B─N peak of BODIPY was observed at 1522 cm⁻¹ (Figure  3a). Conjugation of BODIPY to the star PEtOx polymer was further corroborated by the ¹H NMR spectrum (Figure  4a), with proton signals of BODIPY appearing at 7.45–7.10 ppm, except for the triazole proton formed by click reaction at 7.87 ppm, and aromatic protons belonging to the initiator (7.08–6.61 ppm). Finally, the nanogel was synthesized utilizing N,Nʺ‐(disulfandiylbis(ethane‐2,1‐diyl)) bis (prop‐2‐yn‐1‐amine) (BAC) as a crosslinker through click chemistry in the presence of Cu(I)Br and pentamethyldiethylenetriamine. N,Nʺ‐(disulfanediylbis(ethane‐2,1‐did))bis(prop‐2‐yn‐1‐amine) was employed as the cross‐linker for BODIPY conjugated‐PEtOx nanogel (NG2 nanogel) synthesis.

Figure 2.

Schematic illustration of fabrication and functionalization of multi‐functionalizable PEtOx based nanogel.

Figure 3.

FT‐IR spectra of a) BODIPY‐conjugated PEtOx star‐polymer, b) NG4 nanogel, and c) NG6 nanogel.

Figure 4.

¹H NMR spectra of BODIPY conjugated‐PEtOx star‐polymer, b) NG4 nanogel, and c) NG6 nanogel.

3.2. Characterizations of Nanogel Formulations without Active Ingredients and Active Substances

The nanogels, based on poly(2‐ethyl‐2‐oxazoline), were synthesized in a dilute aqueous medium in the presence of [N,Nʹ‐(disulfandiylbis(ethane‐2,1‐diyl)) bis (prop‐2‐yn‐1‐amine)] cross‐linker containing a disulfide bond to ensure their redox sensitivity. Subsequently, the cross‐linked nanogels, conjugated with BODIPY, were further modified by attaching an alkyne‐containing PSMA peptide. The characterization of the nanogel formation was performed using various techniques, including DLS, NMR spectroscopy, FT‐IR, TEM, and ESEM imaging. The successful synthesis of the nanogels was confirmed through several key observations. Firstly, a new peak emerged at δ = 2.63 ppm (triazole‐CH ₂ ‐NH) in the ¹H NMR spectrum (Figure 4b), signifying the occurrence of nanogel synthesis. Additionally, the FT‐IR spectrum (Figure 3b) revealed a reduction in the peak intensity of ─N₃ at 2109 cm⁻¹ and an increase at 3390 cm⁻¹, both of which are characteristic of nanogel formation.

In the ¹H NMR spectrum (Figure 4b), proton signals corresponding to the polymer were observed at δ = 0.93 ppm, δ = 2.29–2.24 ppm, and δ = 3.40 ppm. Furthermore, signals indicating the presence of the BODIPY‐derived fluorescent dye were detected at δ = 1.12 ppm and δ = 6.79–6.75 ppm. The drug active ingredient candidate, SGK 636, synthesized by our research group,^{[ 28 ]} was successfully encapsulated into the nanogel during the production process, as confirmed by both ¹H NMR and FT‐IR spectra.

The presence of the triazole CH₂─NH peak at 2.63 ppm indicated the occurrence of nanogel synthesis. The signals observed in the ranges of 7.62–6.59, 4.24–4.09, and 1.64–1.52 ppm in the ¹H NMR spectrum belong to the drug's active ingredient candidate. After the formation of the nanogel, a decrease in the peak intensity of ─N₃ at 2109 cm⁻¹ and an increase in the peak intensity at 3390 cm⁻¹ for ─NH and ─OH functional groups were observed in the FT‐IR spectrum (Figure 3b). Furthermore, the presence of BODIPY was once again confirmed by the B─N stretching peak at 1510 cm⁻¹ in the FT‐IR spectrum. For the targeted delivery of the drug‐loaded nanogel to cancer cells, PSMA‐specific peptide 563 was conjugated to the nanogel using click chemistry. The peak observed at 8.06 ppm in the ¹H NMR spectrum (Figure 4c) corresponds to the NH protons of amino acids. Additionally, specific signals from serine (S) at 3.48 ppm, threonine (T) at 1.21 ppm, and leucine (L) and isoleucine (I) amino acid proton signals at ≈0.82 ppm were observed. As shown in the FT‐IR spectrum in Figure 3c, vibrations of NH and OH functional groups of amino acids were observed in the range of 3520–3383 cm⁻¹. The vibration from the azide end group at 2100 cm⁻¹ completely disappeared, indicating the successful conjugation of the peptide to the nanogel.

A spectrofluorometer was utilized to confirm the covalent attachment of the BODIPY dye to the nanogel. As a control, BODIPY‐unbound star‐PETOx polymer was used, and no emission was observed. The absorbance of BODIPY and Peptide 563 attached to the nanogel was measured at 800 nm. The nanogel bound with BODIPY and peptide 563, and loaded with SGK 636, emitted light at 802 nm (Figure  5 ). This demonstrates that the nanogels possess desirable properties in the near‐infrared (NIR) region, making them suitable as macromolecular imaging agents.

Figure 5.

Fluorescence spectra of star‐PEtOx polymer, NG5 nanogel, and NG6 nanogel.

3.3. Particle Size and Morphology of Nanogels

Nanoparticles, which need to circulate in the bloodstream for an extended period, should ideally fall within the size range of 30–200 nm to accumulate in tumor tissues.^{[ 31 ]} Hydrodynamic diameter analysis, particle size distribution, and zeta potentials of the nanogels were measured using dynamic light scattering (DLS) (Table  1 ). The analysis of average particle size revealed that the nanogels indeed fell within the nanometer range. Additionally, it was observed that all nanogels, except for those conjugated with peptide and fluorescent dye, displayed a hydrodynamic diameter below 200 nm. Consequently, nanogels with appropriate particle sizes were successfully prepared to leverage the enhanced permeability and retention (EPR) effect. A reduction in particle size was observed due to the physical interactions between the nanogel and the drug‐active substance. The ζ‐potential values of NG1, NG3, and NG6 nanogels were determined to be −5.02 ± 0.2, −3.14 ± 0.5, and −0.33 ± 0.7 mV, respectively.

Table 1.

Size, PDI, zeta potential, and encapsulation efficiency results of nanogel formulations prepared with star‐PEtOx polymer (n = 3).

Nanogel	Active ingredient	Particle size [nm] a)	Particle size [nm ± SD] b) n = 3	PDI b) n = 3	Zeta potential [mV] ± SD	Encapsulation Eefficiency [% ± SD]
NG1	–	–	142.6 ± 19	0.195	−3.14 ± 0.5	–
NG2	SGK 636	–	136.2 ± 18	0.142	–	68.76 ± 4.2
NG3	–	176.6	194.5 ± 8.8	0.548	−5.02 ± 0.2	–
NG4	SGK 636	115.5	123.7 ± 0.9	0.425	–	67.87 ± 5.2
NG5	–	155.7	233.8 ± 22.4	0.303	–	–
NG6	SGK 636	153.8	161.7 ± 0.9	0.297	−0.33 ± 0.7	64.27 ± 3.6

NG1, PEtOx nanogel (SGK636 free); NG2, SGK636 loaded‐PEtOx nanogel; NG3, BODIPY conjugated‐PEtOx nanogel (SGK636 free); NG4, SGK636 loaded‐BODIPY conjugated‐PEtOx nanogel; NG5, Peptide563 and BODIPY conjugated‐PEtOx nanogel (SGK636 free); NG6, SGK636 loaded‐Peptide563 and BODIPY conjugated‐PEtOx nanogel; SD, standard deviation; PDI, polydispersity index

^a) Statistical dimensions were calculated from SEM images using Imagej software;

^b) With dynamic light scattering.

The formulations exhibited clear nanogels with a uniform particle distribution and consistent dispersion. The shape and surface morphology of the nanogels, prepared under optimized conditions, were examined using environmental scanning electron microscopy (ESEM) and transmission electron microscope (TEM). The study revealed that the nanogel particles exhibited uniform dispersion, were spherical in shape, and had sizes in the nanometric range. Average particle sizes were calculated using particle size distribution histogram plots (Figure  6 ). It is worth noting that there may be differences when comparing the diameter and hydrodynamic size of the nanogels observed in ESEM, TEM, and DLS. ESEM images of the nanogels were captured under dry conditions, while hydrodynamic size measurements were performed in an aqueous medium using DLS and TEM. It should be emphasized that the hydrodynamic size observed in DLS and TEM was larger than that observed in ESEM, indicating that the nanogels swelled in an aqueous solution but shrank under dry conditions.

Figure 6.

Size and morphological characterization of nanogels. Environmental scanning electron microscope (ESEM) images and size distribution (analyzed using ImageJ software) of NG3, NG4, NG5, and NG6 nanogels.

The TEM images of nanogels are presented in Figure  7 , where we observe distinct spherical structures, well‐separated from one another. In terms of size distribution data, there were slight differences between the results obtained from TEM, SEM, and zeta sizer. These minor discrepancies could be attributed to each instrument having different techniques and data analysis methods. Additionally, these differences might arise from measuring nanojels in liquid or dry states or some nanogels possibly aggregating during analysis. In any case, the results of these analyses indicate that this formulation is within the nano‐sized range. It is important to emphasize the significance of particle size in terms of adhesion and interaction with biological cells.^{[ 32 ]} The fact that PEtOx nanogel is in the nanoscale range implies a potential to reduce non‐specific liver uptake^{[ 33 ]} and enhance the drug's actual distribution and bioavailability.

Figure 7.

Transmission electron microscope (TEM) images and histogram graph of NG1 nanogel.

3.4. Physical Stability of Drug‐Loaded Nanogel Formulations under Physiological Conditions

When nanoparticles enter the bloodstream, they tend to attract proteins to their surfaces, resulting in the formation of a “protein corona.”^{[ 34 ]} This protein corona can trigger the body's reticuloendothelial system to identify these nanoparticles as foreign invaders, leading to their rapid removal from the bloodstream.^{[ 35 ]} Therefore, assessing the resistance to protein adsorption is a crucial parameter for evaluating the in vivo stealth ability of nanoparticles.^{[ 36 ]} To investigate this phenomenon, NG2 nanogel was subjected to various conditions, including incubation in distilled water, PBS medium (pH 7.4), and PBS medium (pH 7.4) containing 1–5% fetal bovine serum (FBS). The average size of the nanogels was determined by dynamic light scattering (DLS) at 37 °C at specific time intervals (0, 0.5, 1, 2, and 3 s). The results of this time‐dependent particle size analysis are presented in Figure  8 . It was observed that the size of the nanogels did not significantly increase when they were placed in distilled water over time. This suggests that the nanogels swelled in an aqueous environment as they absorbed liquid into their structure. Moreover, the physical stability of the nanogels was found to be higher in pH 7.4 PBS medium. In environments with low serum protein content, the drug‐loaded nanogel formulations exhibited greater stability compared to media with high serum protein levels. However, significant aggregation was observed in the nanogel formulations that were stored overnight in pH 7.4 PBS containing 1% FBS. All nanogel formulations were found to aggregate in media with high serum protein concentrations. It is important to note that formulations intended for intravenous administration carry the risk of aggregation in the bloodstream. The strong interaction observed between nanogels and proteins was likely due to the high concentration of FBS used. The decrease in particle size observed over time can be attributed to the nanogels collapsing into larger particles, with only dispersed nanosized particles measurable during the analysis.

Figure 8.

Physical stability of NG2 nanogel under physiological conditions.

As a result, it was observed that SGK636 loaded‐PEtOx nanogel (NG2 nanogel) formulations in a pH 7.4 PBS medium containing 1% FBS at 37 °C remained more stable compared to media with high serum protein content. These findings suggest that intravenously administered nanogel formulations can maintain their physical stability while circulating in the bloodstream until they reach their intended target site.

3.5. Encapsulation Efficiency of Nanogel Formulations Containing SGK 636 Active Ingredient

The encapsulation efficiency results of NG4 nanogel and NG6 nanogel were found close to the NG2 nanogel formulation.

A calibration curve was established within the range of 5–100 µg mL⁻¹, exhibiting a high correlation coefficient (R ² = 0.998) as depicted in Figure S12, Supporting Information. The encapsulation efficiency was expressed as the percentage of drug encapsulation efficiency (% EE) and calculated using Equation (1). Each sample's mean value was determined as the average of three measurements, and the results were presented as mean ± SD.

The analysis revealed that the encapsulation efficiency of NG6 nanogel formulation was ≈64.27 ± 3.6% (Table 1).

3.6. Cytotoxicity of Empty and SGK 636‐Loaded Nanogels

The cytotoxic effects of both empty and SGK 636‐loaded nanogels were assessed on human normal prostate epithelial cells (PNT1A) and human prostate carcinoma cells (LNCaP and DU‐145) at various doses. Initially, the toxicity of the empty formulation was evaluated on PNT1A cells within a concentration range of 150–750 µg mL⁻¹ (as shown in Figure  9 ). The results revealed that concentrations of 150 and 200 µg mL⁻¹ of the empty nanogel did not lead to a statistically significant decrease in cell viability, with viabilities of 75% and 68%, respectively. However, as the doses increased from 300 to 750 µg mL⁻¹, a significant dose‐dependent reduction in PNT1A cell viability was observed at 72 h, with viability percentages of 28%, 16%, 13%, 16%, and 7%, respectively.

Figure 9.

Cytotoxic effect of the empty nanogel on PNT1A cells. Cells were seeded in 96‐well plates (5 × 10³ cells per well) and treated with 150 to 750 µg mL⁻¹ of the empty nanogel for 72 h. Cell viability was determined using WST‐1 cell proliferation assay. The viability of untreated cells (0 µg mL⁻¹) was considered 100%. The results represent the average of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

These findings suggest that concentrations of up to 200 µg mL⁻¹ of the empty nanogel formulation are not toxic to PNT1A cells, while higher doses result in a substantial decrease in cell viability, reducing it by up to 90%. Consequently, concentrations of up to 200 µg mL⁻¹ of the empty nanogel can be considered safe for use as a drug carrier in subsequent experiments and for cancer therapy.

To assess the impact of SGK 636‐loaded nanogel on established prostate cancer cell lines, two different cell models were selected:^{[ 37 ]} androgen‐responsive LNCaP cells derived from lymph node metastasis^{[ 38 ]} and androgen non‐responsive DU‐145 cells derived from central nervous system metastasis.^{[ 39 ]} A dose‐ and time‐dependent assays were conducted, as depicted in Figure  10 . The results of the 24 h treatment with SGK 636 loaded‐nanogel (NG2 nanogel) concentrations ranging from 10 to 25 µg mL⁻¹ indicated that there was no significant effect on the viability of LNCaP cells. However, following 48 h of treatment, a notable reduction in cell viability by ≈50% was observed in LNCaP cells treated with 25 µg mL⁻¹ of NG2 nanogel. Furthermore, after 72 h of treatment with 17 µg mL⁻¹ of NG2 nanogel, LNCaP cell viability decreased by 70%, indicating a cytotoxic effect. At the higher concentration of 25 µg mL⁻¹, the cytotoxic effect was even more pronounced, resulting in an 85% reduction in viability. In contrast, for DU‐145 cells, neither of the tested doses of NG2 nanogel significantly reduced cell viability below 100% at 24 and 48 h. However, after 72 h, concentrations above 200 µg mL⁻¹ of the formulation exerted significant cytotoxicity on DU‐145 cells, leading to a 65–70% reduction in cell viability. Overall, these cell viability results demonstrate that NG2 nanogel treatment leads to a decrease in the viability of both LNCaP and DU‐145 prostate cancer cells, with varying degrees of cytotoxicity observed after 72 h of treatment.

Figure 10.

Cytotoxic effect of the SGK 636‐loaded nanogel (NG2 nanogel) for LNCaP and DU‐145 cells. The cells were seeded in 96‐well plates (5 × 10³ cells per well). A) LNCaP cells were treated with 10 to 25 µg mL⁻¹ and B) DU‐145 cells were treated with 20 to 350 µg mL⁻¹ of NG2 nanogel for 24, 48, and 72 h. Cell viability was assessed using WST‐1 cell proliferation assay at each 24 h interval until 72 h. The viability of untreated cells (0 µg mL⁻¹) was considered 100%. The results represent the average of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3.7. Targeting Efficiency of Peptide 563 and BODIPY‐Conjugated Nanogels (NG6 nanogel) for PNT1A, DU‐145, and LNCaP Cells

To enhance the tumor selectivity of the nanogels, we employed a prostate‐specific membrane antigen (PSMA)‐specific targeting approach in this study. PSMA is a type II transmembrane glycoprotein that is overexpressed in prostate cancer,^{[ 40} , ^{41 ]} PSMA serves as a unique cell surface marker that is negatively regulated by androgen and is often used to identify hormone‐resistant carcinomas and metastatic foci.^{[ 42 ]} The expression of PSMA in prostate cancer cells facilitates the efficient uptake of PSMA‐targeting ligands, reducing the side effects associated with conventional therapies.^{[ 41 ]} Peptide 563 is a tumor‐homing peptide known for its high selectivity for PSMA‐positive prostate cancer cells.^{[ 43} , ^{44 ]}

To assess the targeting specificity of NG5 nanogel (peptide 563 conjugated nanogel) for prostate cancer cells, we evaluated the binding affinity of the NG3 nanogel (without peptide) construct on LNCaP and DU‐145 prostate cancer cells, comparing it to PNT1A normal prostate epithelial cells using flow cytometry. The percentage of affinity for the formulation in each cell type is depicted in histogram plots in Figure  11 . The targeting efficiency of NG5 nanogel for PNT1A cells was 1.04%, while DU‐145 and LNCaP cells exhibited 1.67% and 61.98% efficiency, respectively (Figure 11). In other words, NG5 nanogels demonstrated significantly higher affinity for LNCaP cells compared to PNT1A cells, although their affinity for DU‐145 cells was lower. This observed selectivity of the NG5 nanogel for LNCaP cells aligns with earlier studies reporting the effective binding of peptide 563 to PSMA on the cell surface.^{[ 44 ]} The difference in PSMA expression levels on the surface of LNCaP and DU‐145 cells explains the lower affinity of the NG5 nanogel for DU‐145 cells.^{[ 45 ]} Our findings suggest that targeting tumors using peptide 563‐conjugated nanogels could be a promising approach in prostate cancer treatment.

Figure 11.

Targeting efficiency of NG5 nanogel for PNT1A, DU‐145, and LNCaP cells as determined by flow cytometry. The cells were treated with 200 µg mL⁻¹ of NG5 nanogels for 6 h and fixed with 0.05% formaldehyde. Black solid‐filled plots represent the treated cell populations. Black lines indicate non‐treated cells. The histograms are representatives of three independent experiments.

4. Conclusion

In our study, we synthesized a new PETOx‐based nanogel and successfully incorporated the candidate drug into it. Using the residual azide functional group present in this nanogel, we were able to apply click chemistry to combine the PSMA peptide and the alkyne group containing a fluorescent dye called BODIPY. The nanogel worked with an impressive efficiency of up to 65% to load the active ingredient of the candidate drug. Notably, cytotoxicity tests showed that these nanogels showed no toxicity at concentrations up to 200 µg mL⁻¹. Furthermore, experiments on LNCaP and DU‐145 prostate cancer cells showed that SGK 636‐loaded nanogel reduced viability in these cells, thus indicating the potential therapeutic efficacy of the nanogel. Cellular uptake experiments revealed that the amount of internalization of the nanogel conjugated with peptide 563 by tumor cells was approximately 60 times higher compared to the nanogel without peptide. These results show that a successful PETOx‐based nanogel has been prepared, specifically targeting prostate cancer cells, and that this nanogel has great potential for use in cancer treatment. Such nanoplatforms hold promise for future treatments by providing more effective and targeted drug delivery in cancer treatment.

Conflict of Interest

The authors declare no conflict of interest.

Supporting information

Supporting Information

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.