Engineered urolithin A-laden functional polymer-lipid hybrid nanoparticles prevent cisplatin-induced proximal tubular injury in vitro

Abstract

Functional polymer-lipid hybrid nanoparticles (H-NPs) are a promising class of nanocarriers that combine the benefits of polymer and lipid nanoparticles, offering biocompatibility, structural stability, high loading capacity, and, most importantly, superior surface functionalization. Here, we report the synthesis and design of highly functional H-NPs with specificity toward the transferrin receptor (TfR), using a small molecule ligand, gambogic acid (GA). A fluorescence study revealed the molecular orientation of H-NPs, where the lipid-dense core is surrounded by a polymer exterior, functionalized with GA. Urolithin A, an immunomodulator and anti-inflammatory agent, served as a model drug-like compound to prepare H-NPs via traditional emulsion-based techniques, where H-NPs led to smaller particles (132 nm) and superior entrapment efficiencies (70% at 10% drug loading) compared to GA-conjugated polymeric nanoparticles (P-NPs) (157 nm and 52% entrapment efficiency) and solid lipid nanoparticles (L-NPs) (186 nm and 29% entrapment efficiency). H-NPs showed superior intracellular accumulation compared to individual NPs using human small intestinal epithelial (FHs 74) cells. The in vitro efficacy was demonstrated by flow cytometry analysis, in which UA-laden H-NPs showed excellent anti-inflammatory properties in cisplatin-induced injury in healthy human proximal tubular cell (HK2) model by decreasing the TLR4, NF-κβ, and IL-β expression. This preliminary work highlights the potential of H-NPs as a novel functional polymer-lipid drug delivery system, establishing the foundation for future research on its therapeutic potential in addressing chemotherapy-induced acute kidney injury in cancer patients.

Graphical Abstract:

1. Introduction

Over the years, nanomedicines have gained growing interest for their ability to customize release profiles, diminish side effects and safeguard the drugs and therapeutics against degradation [1–5]. Among various types of nanoparticles such as drug-laden polymer or lipid nanoparticles, liposomes, polymer-drug conjugates, polymersomes, carbon nanotubes and gold nanoparticles, lipid- and polymeric-based nanoparticles stand out as two extensively studied and promising classes of drug delivery systems [6]. Both techniques have outstanding biocompatibility and biodegradability [7], lipid nanoparticles (L-NPs) have the capacity to encapsulate and transport both hydrophobic and hydrophilic drugs effectively [8], facile preparation through methods that are free from organic solvents, and straightforward scalability [9–11]. On the other hand, polymeric nanoparticles (P-NPs) offer their own set of advantages that they are easily fabricated, non-toxic, non-immunogenic, and have a wide diversity in structure and functionality, allowing for easier and more efficient functionalization for targeted drug delivery [12–15]. Despite notable advancements in designing nanoparticle carrier systems with enhanced functionality, specificity, and disease-targeting capabilities, only a handful of drugs based on lipids/protein have reached the market, e.g., Doxil^®, AmBisome^®, Amphotech^®, Epaxel^®, Abraxane^®, Onpattro^®, and certain Covid-19 vaccines. While there could be multiple explanations; limited market presence in parts could be attributed to the choice of drug and the corresponding disease. The type of target, route of administration, limited efficacy of active systems due to lack of functionality compared to passive systems, poorly established risk-benefit profiles, economic viability, and generalizing based on size without considering the carrier material and drug [16,17]. In line with lipids, conventional polymers are also limited by their functionality, drug payload, and choice of ligands [18–20].

Very recently, there has been a significant drive-in excipients research to meet the challenges posed by the new drug discovery pipeline [21,22]. In this context, hybrid nanoparticles like (a) polymers physically adsorbed on the surface of L-NPs, (b) polymers trapping lipid vesicles, (c) polymers covalently bonded to the polar head of phospholipid and (d) amphiphilic polymers homogenously distributed with L-NPs monomers forming a network around lipid vesicles are finding applications in drug delivery [23–27]. However, these hybrid particle architectures still need to be more adequately reported, demanding a more profound comprehension of their preparation methods, physicochemical properties, and biological behavior.

Here, we report a novel hybrid nanoparticle (H-NPs), constituted of a core made up of glyceryl tristearate, a triglyceride, while the surface is comprised of a polymer functionalized with gambogic acid (P2s-GA). The polymer (P2s-GA) promotes transferrin receptor interaction, facilitating transcytosis across the intestinal barriers upon oral administration [28–31]. The H-NPs combine the advantages of both L-NPs and P-NPs, enabling the development of oral drug products [32]. To demonstrate the feasibility of H-NPs as a delivery vehicle, we have used Urolithin A (UA) as a model compound. UA is a naturally occurring gut metabolite of ellagic acid known for its intriguing anti-inflammatory and antioxidant properties. It enhances cellular health by promoting mitophagy and mitochondrial function while mitigating harmful inflammation [33]. However, its clinical application is hindered by its poor oral bioavailability [32–35]. We have conducted an in vitro efficacy study in cisplatin-induced injury in healthy human proximal tubular cells (HK2), where H-NPs offered superior anti-inflammatory properties compared to individual NPs. Further studies in whole body systems, e.g., cancer models, are needed to validate the use of H-NPs in preventing chemotherapy-induced kidney injury without compromising the chemotherapeutic efficacy.

2. Materials and Methods

2.1. Materials

Glyceryl tristearate (tristearin), D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS), N-boc ethylenediamine, Sn(Oct)₂ (Sigma-Aldrich) and coumarin were purchased from Sigma-Aldrich, Merck (Darmstadt, Germany). Gambogic acid (GA) was obtained from BroadPharm, UA was purchased from Angene (London, UK), PEG Mn = 380 – 420 (EMD Chemicals), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride (Oakwood Chemicals), cyclohexanetetracarboxylic dianhydride (HCDA) (Ambeed) and DMSO-d6 (MagniSolv) - solvent for NMR. HPLC solvents and all other chemicals were of analytical grade and were purchased from Fisher Scientific (USA).

2.2. Methods

2.2.1. Polymer Synthesis (P2s-GA and Rh-P2s-GA):

We have used periodic functional polyester, developed in our lab that offers better functionality compared to conventional polyesters [28,35]. In brief, we prepared a PLA-PEG-PLA triblock copolymer, which was further reacted with cyclohexanetetracarboxylic dianhydride (HCDA), imparting periodically spaced carboxyl groups for GA conjugation via ethylenediamine linker using EDC chemistry. The GA-conjugated polymer (P2s-GA) was thoroughly characterized using NMR and GPC for successful conjugation and molecular weight, respectively (Figure S1). A fluorescently labeled polymer (Rh-P2s) was synthesized by adding Rhodamine (Rh) instead of GA in a similar fashion.

2.2.2: UA-laden and Fluorescent Nanoparticle Preparations

(a) P-NPs [P-NPs (UA) and Rh-P-NPs] were fabricated using the emulsion-diffusion-evaporation technique. In brief, P2s-GA (50 mg) and 15 mg of P2s (to compensate for the polymeric backbone loss due to GA mass) were dissolved in 2 mL of ethyl acetate. Simultaneously, 5 mg of UA (10% w/w) was dissolved in 100 μL of DMSO and 0.5 mL of DCM while stirring for 1 hour. Subsequently, the UA and polymer solutions were mixed and stirred for an additional 30 minutes as the organic phase. For the aqueous phase, 50 mg of TPGS (1% w/w) was dissolved. The organic phase was then added dropwise to the aqueous phase while stirring at 1400 rpm for 6 minutes. This emulsion was followed by homogenization at 15000 rpm for 6 minutes (Kinematica). The final emulsion was added to 10 mL of water and stirred for 3 hours to ensure complete evaporation of the organic solvent. The nanoemulsion was centrifuged at 15000 g for 30 min at 4°C to separate the pellet of formulation from the supernatant. To prepare labeled Rh-P-NPs, 2 mg of Rh-P2s (4% w/w) was added to P2s-GA (50 mg), dissolved in 2 mL of ethyl acetate, and 0.5 mL of DCM to form the organic phase. The subsequent steps in the preparation remained the same as described above.

(b) L-NPs [L-NPs (UA) and L-NPs (C6)] were synthesized using a solvent evaporation technique based on emulsification followed by homogenization and ultrasonication. The emulsion was composed of a TPGS (1% w/w) aqueous solution as the dispersing phase and tristearin (1% w/w) as the disperse phase. In summary, 50 mg of tristearin was dissolved in 2.5 mL of ethyl acetate at 80 °C. The resulting lipid solution was added to the TPGS aqueous solution and homogenized at 15,000 rpm at 80 °C for 1 min. The emulsion was further homogenized through ultrasonication (30% amplitude for 45 sec). The final dispersion was diluted with 10 mL of water and stirred for 3 h. L-NPs were then recovered by centrifugation (5000 rpm for 20 minutes at 25°C). For the preparation of fluorescent-labeled L-NPs [L-NPs (C6)], Coumarin (C6) (0.5% w/w, with respect to the entire weight of the lipid) was added to the lipid phase, while the remaining steps in the preparation were consistent with the description above. UA-loaded L-NPs [L-NPs (UA)] were obtained by incorporating 2.5 mg (5% w/w) of Urolithin A, previously solubilized in 100 μL of DMSO, into the lipid phase before the emulsification step.

(c) H-NPs [H-NPs (UA) and Rh-H-NPs (C6)] were prepared following emulsion-diffusion-evaporation technique, based on ultrasonication and homogenization. Briefly, P2s-GA (25 mg) and tristearin (25mg) were dissolved in ethyl acetate (2 mL), and 0.5ml of DCM with 5 mg of UA (10% w/w) separately dissolved in 100 μl of DMSO and emulsified in 5 mL TPGS (1% w/w, with respect to the whole weight of the dispersion) aqueous solution while stirring at 1400 rpm. The emulsion was further stirred at 1400 rpm for 6 min, followed by ultrasonication (30% amplitude for 45 sec). The final dispersion was diluted with 10 mL water and stirred for 3 h. H-NPs were recovered by 2 cycles of centrifugation (1^st: 5000 rpm for 20 min; 2^nd: 8000 rpm for 20 min). To prepare fluorescent labeled Rh-H-NPs (C6), 1 mg of P2s-Rh (4% w/w) and C6 (0.5% w/w of lipid) were added to the organic phase while the rest of the steps in the preparation remained the same as described above.

2.2.3: Lyophilization

P-NPs (UA), L-NPs (UA), H-NPs (UA), Rh-P-NPs, L-NPs (C6), and Rh-H-NPs (C6) pellets obtained after centrifugation were suspended in 1 mL of trehalose dehydrate solution (5% w/w) and subjected to freezing at −80 °C followed by primary drying (−55 °C/0.004 mbar for 48 h) and secondary drying (20°C/0.004 mbar for 24 h). The lyophilized samples were crimped and stored at 4°C till further use.

2.2.4: Characterization

(a) Size and Zeta potential: Dynamic light scattering (DLS) analysis was conducted at 25°C using a Malvern Zetasizer Nano-ZS. For the analysis, 2 μL aliquots of the bulk nanoparticle suspensions were diluted with 1 mL of distilled, deionized (DDI) water for size measurements.

(b) Morphology: Aliquots of 2 μL of P-NPs (UA), L-NPs (UA), and H-NPs (UA)of a concentration of 1 μg/ml of UA equivalent were deposited on a silica wafer. The samples were carbon coated before visualization and scanned using an Apreo FE-SEM, operating at a working distance of 5–7 mm and 5 kV.

(c) Entrapment Efficiency: To evaluate entrapment efficiency, each formulation was subjected to a specific cycle of centrifugation, as mentioned under preparation section 2.2.2 (a-c), to separate the dispersant phase from UA-loaded nanoparticle pellets. UA loaded P-NPs, L-NPs, and H-NPs pellets were dissolved in acetonitrile: methanol (50:50). UA concentration was determined for both dissolved pellets solution and dispersant phase, to evaluate the percentage of loaded UA and free UA respectively. Entrapment Efficiency was determined for each formulation using the following equation:

$$\text{Entrapment}\text{Efficiency}=\text{UA}/{\text{T}}_{\text{UA}}\times 100$$

in which UA is the amount of drug detected in the dissolved pellets solution and T_UA is the total amount of drug used for nanoparticles production.

UA quantification via HPLC was performed on an Agilent 1260 Infinity LC with a diode array detector (DAD). The LC was fitted with a guard column in series with an Agilent Poroshell 120 C-18 column (100 mm × 4.6 mm × 2.7 μm), operating at a flow rate of 1.0 mL/min, at 60 °C and monitoring a wavelength of 280 nm. The mobile phase consisted of 95:5 (A: B) where A was acetonitrile and B was methanol with 1% formic acid. The UA concentration was calculated from a standard calibration curve (in μg/mL).

(d) Structural Characterization: Structural alignment of all of formulations P-NPs, L-NPs and H-NPs were performed via confocal laser scanning microscopy (CLSM) analysis. Fluorescently labeled formulations [Rh-P-NPs, L-NPs (C6), Rh-H-NPs (C6)] were suspended in deionized water, smeared on a glass coverslip, dried, and imaged under confocal microscopy (Zeiss LSM-900) (40x) with digital magnification set at 35 and DIC channel for surface observation. Image analysis was performed using ZEN Blue software.

2.2.5: In Vitro Analysis

(a) Cellular uptake and viability using human normal intestinal cell line (FHs 74): To investigate cellular uptake, FHs 74 cells suspension 50000 cells per sample (passage 8–11) were incubated with 50 μg/mL of fluorescent-labeled formulations [Rh-P-NPs, L-NPs (C6), Rh-H-NPs (C6)] suspended in nano-distilled water for 1 hour at 37°C/5% CO₂. After incubation, the medium was replaced with fresh FACS buffer, and the cells were washed three times to remove unbound and non-internalized nanoparticles by centrifuging at 700 g for 6min. The final cell suspension was treated with 3 μL of 7-aminoactinomycin D (7-AAD) for cell viability and subjected to flow cytometry (FACS) analysis using Attune NxT flow cytometer. FACS results were analyzed using FCS Express 7 Research Edition software.

For cell viability analysis in HK2 and FHs 74 cells, cell suspensions containing 1 × 10⁵ cells were incubated with 50 μg/mL of the fluorescent-labeled formulations (re-suspended in nano-distilled water) for 1 h at 37° C/5% CO₂. After treatment, cell suspensions were centrifuged, washed three times, and resuspended in FACS buffer (PBS with 1% BSA). The final cell suspension was treated with 3 μL of 7-AAD and subjected to FACS analysis using Attune NxT flow cytometer. Untreated cells were included as a control for all analyses, and UltraComp eBeads^™ compensation beads from Invitrogen were used as compensation controls for nanoparticles. FACS results were analyzed using FCS Express 7 Research Edition software.

(b) Efficacy of UA-laden nanoparticles [H-NPs (UA), L-NPs (UA), and P-NPs (UA)] in HK2 cells challenged with cisplatin: In this study, HK2 cells were cultured in Dulbecco’s Modified Eagle Medium F12 (DMEM-F12) medium, utilizing cells from passages 9–12. Following trypsinization, cell suspensions containing 1 × 10⁵ cells were incubated with 20 μM cisplatin for 6 hours and subsequently treated with UA-laden nanoparticles at concentrations of 10 μM, equivalent to the concentration of UA. After the treatment period, Eppendorf tubes underwent centrifugation at 700 g for 6 min, leading to the removal of the supernatant followed by resuspension of the cell pellet in flow buffer (200–300 μL). The subsequent 45 min incubation with labeled primary antibodies targeting TLR4, NF-κB, and IL-1β was followed by repeated centrifugation and resuspension steps. The final step involved the addition of 3 μL of 7-AAD per sample, culminating in FACS analysis using FCS Express 7 Research Edition software.

(c) Confocal microscopy of FHs 74 Int and HK2 cells: FHs 74 and HK2 cells were cultured in 8-well glass-bottom plates at a density of 5,000 cells per well. FHs 74 cells were maintained Hybri-Care Medium ATCC 46-X supplemented with 30 ng/ml epidermal growth factor (EGF), fetal bovine serum (FBS), 10%,1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. HK2 Cells were maintained in DMEM-F12 medium, supplemented with 10% FBS and 1% penicillin-streptomycin, at 37°C in a humidified atmosphere containing 5% CO₂. After 24 h of seeding, the cells were treated with three different nanoparticle formulations: coumarin-labeled nanoparticles (L-NPs (C6)), rhodamine-labeled nanoparticles (Rh-P-NPs), and a combination of both (Rh-H-NPs (C6)). Each treatment was administered at a concentration of 5 μg/ml and allowed to incubate for 1 h.

Following nanoparticle treatment, cells were washed three times with phosphate-buffered saline (PBS) to remove unbound particles. For cells treated with Rh-P-NPs and Rh-H-NPs (C6), ActinGreen 488 ReadyProbes Reagent was used to stain the actin filaments, and Hoechst dye was used for nuclear staining. In contrast, cells treated with L-NPs (C6) were stained with Actin Red 555 ReadyProbes Reagent for actin visualization alongside Hoechst for nuclear staining. Staining was performed according to the manufacturer’s instructions, with cells incubated in the dark at room temperature for 1 h.

Post-staining, cells were fixed with 2% paraformaldehyde for 30 min at room temperature. Following fixation, cells were washed three times with PBS to remove excess paraformaldehyde.

Fixed and stained cells were imaged using a LSM900 laser scanning confocal microscope equipped with appropriate filters for the detection of coumarin, rhodamine, ActinGreen 488, Actin Red 555, and Hoechst signals. Images were captured using a 40x oil immersion objective to ensure high-resolution visualization of the nanoparticles and cellular structures. Image analysis was performed using ZEN blue software 3.4.

2.2.6: Statistical Analysis:

For the cell uptake studies, Student’s t-test was employed. The sample sizes ranged from 6 to 8 replicates for each condition, ensuring robust statistical power to detect significant differences. For flow cytometric analysis, quadruplicates were utilized for all samples, and a two-way ANOVA was performed to analyze data. Following the two-way ANOVA, post hoc tests, such as Tukey’s multiple comparisons test, were conducted using GraphPad Prism software version 10.0.2. Throughout the analyses, a significance threshold of p<0.05 was applied to determine statistically significant results.

3. Results and Discussion

3.1. Polymer Synthesis:

P2s polymer, after conjugation with GA, gave a yellow powder, P2s-GA. The ¹H nuclear magnetic resonance (NMR) spectra of P2s-GA revealed the PLA methyl and methylene proton peaks at 1.55 ppm and 5.11 to 5.15 ppm, respectively, and PEG methylene at 3.60 to 3.62 ppm. The PEG methylene linkage to the carbonyl ester ends of PLA was confirmed by the resonance signals at 3.65 and 4.20 to 4.30 ppm, while the signal at 8 ppm is due to the presence of amide bond, and doublets at 5.5–6 ppm are due to GA (Figure S1).

3.2. UA-laden or Fluorescent Nanoparticles:

P-NPs (UA), L-NPs (UA), and H-NPs (UA) were formulated using three distinct solvent evaporation techniques due to the nature of the excipients used, with formulation details outlined in Table 1. As mentioned earlier, the P-NPs were obtained through a highly reproducible method that had been thoroughly optimized in our laboratory [28]. Tristearin was chosen as the solid lipid component for both L-NPs and H-NPs due to its well-established properties, including biocompatibility, biodegradability, and its ability to form mono-component solid lipid nanoparticle systems without the need for an additional lipidic component [36,37]. While we achieved higher UA loading in both H-NPs and P-NPs, the loading capacities were limited in the case of L-NPs and could not do beyond 5%.

Table 1:

Formulation Details of nanoparticles prepared for the study.

S.No.	Formulation	Size (nm)	Entrapment Efficiency (%)	Zeta Potential (mV)	PDI
1.	P-NPs (UA)	157.40±22	52.30±0.26	9.48±0.59	0.26±0.03
2.	H-NPs (UA)	132.57±3	70.07±8.69	9.96±1.23	0.26±0.04
3.	L-NPs (UA)	186.17±8	29.47±3.33	−11.27±0.92	0.28±0.02
4.	Rh-P-NPs	155.23±41	-	26.40±0.95	0.24±0.01
5.	Rh-H-NPs(C6)	184.50±31	-	24.20±0.26	0.24±0.01
6.	L-NPs(C6)	190.90±16	-	−22.17±1.00	0.28±0.02

3.3. Lyophilization:

An often-overlooked aspect in the literature is the determination of optimal storage conditions for lipid nanoparticles. The efficacy of lipid-loaded drugs can exhibit variations at different temperatures, ranging from −80 to 40°C, making reproducibility a significant concern [38,39]. Apart from the degradation of the drug itself and the particles growth as a result of Ostwald ripening processes [40], unoptimized storage conditions may also lead to particles aggregation and/or lipid crystallization or polymorphic transformations resulting in drug expulsion [41]. To address this, we employed lyophilization for all formulations before in vitro experiments to ensure uniformity in excipient behavior. The freeze-dried formulations displayed a cake-like appearance (Figure S1), a crucial consideration in design and development to enhance the shelf-life of drugs.

3.4. Characterization:

Upon increasing the loading from 5% to 10% w/w, the L-NPs (UA) formulation experienced phase separation immediately after preparation, likely attributed to structural overloading and subsequent loss of integrity. H-NPs not only exhibited comparable loading capacities but also demonstrated superior entrapment efficiency (Table 1), surpassing both L-NPs and P-NPs. All variations of the nanoparticles prepared were less than 200 nm and displayed a spherical shape, characteristics favorable for oral absorption (Figure 1) [42]. Zeta-potential values obtained via Dynamic Light Scattering (DLS) were consistently above ±10–20 eV at pH 6–7 for all formulations, indicating a moderate degree of repulsion between nanoparticles in the disperse phase and implying the stability of the nanoparticles. Notably, the zeta potential value was approximately +25 eV for both P-NPs and H-NPs, whereas, for L-NPs, it assumed a negative value around −22 eV due to the presence of negatively charged polar groups of tristearin on the L-NPs surface. This suggests the formation of hybrid nanoparticles characterized by a polymeric-dense surface.

Figure 1:

The particle characteristics of UA-laden HNPs (a-c) Scanning electron micrographs depicting spherical morphology and (d-f) Representative dynamic light scattering (DLS) plots showing z-average size distribution by intensity

CLSM microscopy analysis was performed on fluorescently labeled formulations to investigate their structural architecture and verify the formation of the desired lipid-rich core/polymer-rich outer layer H-NPs [43,44]. CLSM visualization of L-NPs (C6), Rh-P-NPs, and Rh-H-NPs (C6) (Figure 2a–c) indicated moderate agreement with SEM and DLS values. The Z-stacking analysis highlights the presence of a heterogeneous spatial distribution of the polymer forming a boundary/superficial region characterized by a rhodamine emission intensity significantly higher than that of the core region where the coumarin intensity was high (Figure 2d), confirming the formation of a polymer-rich outer layer surrounding a lipid-rich core revealing the high lipophilicity of the core, probably due to the formation of the nanocomposites H-NPs from lipid-dense aggregation nuclei. The emission spectra Rh-H-NPs (C6) confirmed the presence of both rhodamine and coumarin (Figure 2e–f and S2) and are quantified (Figure 2g).

Figure 2:

The core-shell framework structure of HNPs analyzed by confocal microscope. (a) Rh-P-NPs; (b) L-NPs (C6);(c) Rh-H-NPs(C6); (d) Z-stacking of Rh-H-NPs(C6) with 28 slices for a 2.5-micron thickness to visualize the internal core (Coumarin 6) and the outer layer (Rhodamine); (e) Rhodamine emission spectra of labeled formulations; (f) Coumarin Emission Spectra of labeled formulations and (g) Quantification of MFI’s of different labeled formulations. Pictures of labeled formulations were obtained using an immersion oil objective (40x) with digital magnification set at 35X and a DIC channel for surface observation.

3.5. In-vitro Studies:

(a). Cellular Uptake and Viability:

The long-term goal is to develop H-NPs as a potential oral delivery platform for poorly soluble and permeable drugs, and their use in treating chemotherapy-induced kidney injury. In this context, FHs 74 cells were selected as a model for intestinal uptake to evaluate and compare the uptake of the formulated nanoparticles, whereas HK2 cells were chosen as a model due to the pivotal functions of the kidney in metabolism and excretion, as well as its susceptibility to damage from chemotherapy drugs.

As the particle size of all three variations is less than 200 nm, this indicates internalization via clathrin and caveolae-mediated pathways involving endocytosis [45]. The qualitative visualization and quantification of the nanoparticle internalization via CLSM/FACS is shown in Figures 3a–c, S3 and S4 respectively. CLSM results are in partial agreement with FACS data, displaying a higher intracellular and superficial accumulation of Rh-H-NPs (C6) with respect to Rh-P-NPs as highlighted also by MFI’s values obtained via CLSM analysis (Figure 3d–e). However, the large MFI difference estimated by CLSM and FACS could be due to the difference in the nature of experiments, the volume of the cells, and particle concentrations. Another thing in both data sets is that L-NPs (C6) have the least MFI in comparison to Rh-P-NPs and Rh-H-NPs (C6), suggesting the role of GA in TfR-mediated active transport, as opposed to passive L-NPs.

Figure 3:

Cell uptake of nanoparticles as a function of surface characteristics using normal intestinal cells (FHs 74 Int). (a) Rh-P-NPs; (b) L-NPs(C6); (c) Rh-H-PNs(C6); quantification of mean fluorescence intensity (MFI) using (d) ZEN Blue software (for a group of 6 images) in CLMS. CLSM analysis of FHs 74 cells treated with 50 μg/ml concentration of different nanoparticles for cellular internalization and membrane visualization was performed using contrast vibrant membrane actin stains. Nuclei were stained with a Hoechst 33342 stain. (Statistical analysis was carried out using student’s t-test (**** P < 0.005 for comparisons between Rh-P-NPs and Rh-H-NPs(C6) ****P < 0.005 and (e) flow cytometric analysis of nanoparticle association with cells. (Statistical analysis was carried out using two-way ANOVA. (****P < 0.0001 for comparisons between Rh-P-NPs and Rh-H-NPs(C6) **** P < 0.0001 for comparisons between L-NPs(C6) and Rh-H-NPs(C6).

Regarding HK2 cell uptake, the qualitative visualization of the nanoparticle’s internalization via CLSM is shown in Figures 4a–c, S5 and S6. MFI values obtained via CLSM analysis (Figure 4d) and FACS analysis (Figure 4e) highlight the same trend obtained for FHs74 cells. No statistically significant differences were observed for Rh-P-NPs and Rh-H-NPs (C6), while L-NPs (C6) showed the lowest uptake with an almost 2-fold decreased MFI’s value. To assess the in vitro cytotoxic/apoptotic effects of the formulations against both FHs 74 and HK2 cells, FACS analysis was employed using 7AAD to discriminate between live and dead cells. No relevant differences in the viability for both FHs 74 Int and HK2 cells were detected between control untreated cells and nanoparticles-treated cells (Figure S3 and S5).

Figure 4:

Cell uptake of nanoparticles as a function of surface characteristics using normal kidney cells (HK2). (a) Rh-P-NPs; (b) L-NPs(C6); (c) Rh-H-PNs(C6); quantification of mean fluorescence intensity (MFI) using (d) ZEN Blue software (for a group of 6 images). CLSM analysis of HK2 cells treated with 50 μg/ml concentration of different nanoparticles for cellular internalization and membrane visualization was performed using contrast vibrant membrane actin stains. Nuclei were stained with a Hoechst 33342 stain, and (e) Flow cytometric analysis of nanoparticle association with cells. (Statistical analysis was carried out using student’s t-test and two-way ANOVA. (**** P < 0.0001 for comparisons between Rh-P-NPs and Rh-H-NPs(C6)

In addition, the relevance of CD71 in both FHs 74 and HK2 cells receptor-mediated uptake of P-NPs and H-NPs was evaluated via FACS analysis. FHs 74 and HK2 cells were labeled with anti-CD71 fluorescent PE (phycoerythrin)–Cy7 antibody, and after treatment with each nanoparticle, the CD71 vs. Rh/C6 density plot (Figure S2 and S4) were measured, and percentage positive cells values extrapolated (Figure S3d and S5d). Consistent with our prior findings, the surface conjugation of GA leads to nanoparticles with a notable affinity for Tfr, resulting in an effective non-competitive receptor-mediated active cellular transport [29]. The percentage CD71 positive cells value, expressing the magnitude of bounded CD71 receptor, was negligible for L-NPs (C6) and was above 60% for both Rh-P-NPs and Rh-H-NPs (C6) in the case of FHs 74 cells and slightly lower (~40%) for HK2 cells.

(b). UA-laden Nanoparticles Effects in Cisplatin-treated HK2 Cells:

The concluding section of the research, focused on evaluating the impact of UA-laden nanoparticles on HK2 cells challenged with cisplatin. The objective was to investigate the potential therapeutic benefits of this formulation in alleviating cisplatin-induced acute kidney injury (AKI). Cisplatin, widely used as an antineoplastic drug for various carcinomas and sarcomas, is known to induce several severe side effects, including AKI. This condition is characterized by tubulointerstitial inflammation and interstitial nephritis.

Recent evidence suggests that proinflammatory and oxidative stress pathways play a crucial role in the pathogenesis of AKI [46]. Consequently, the regulation of these pathways may offer protective effects [47]. In our laboratory, previous research has demonstrated the mitigating effects of UA in polymeric nanoparticles (P-NPs (UA)) in reducing cisplatin-induced intracellular inflammatory and apoptotic mechanisms in a rat model [48]. Literature supports the involvement of the TLR4/NF‐κB pathway in inflammation [49–51]. Moreover, TLR4 activation has been shown to trigger the release of inflammatory cytokines, such as IL-1β, mediated by NF‐κB [52,53]. This information suggests a potential mechanism through which UA-loaded nanoparticles may exert their protective effects by modulating the TLR4/NF‐κB pathway and reducing inflammatory responses in the context of AKI induced by cisplatin.

The downregulation of TLR4, NF- κB, and IL-1β expression were evaluated via FACS analysis. The administration of cisplatin (CIS) to HK2 cells significantly increased the expression of proinflammatory markers, with TLR4, NF-κB, and IL-1β levels rising by 5-, 8-, and 3-fold, respectively, when compared to the healthy control group. This elevation confirmed the proinflammatory impact of CIS on HK2 cells (Figure 5, S7). Notably, the pro-inflammatory response elicited by CIS was effectively mitigated when cells were co-incubated with various UA formulations, including plain UA, L-NPs (UA), P-NPs (UA), and H-NPs (UA), all at a concentration of 10 μM.

Figure 5:

In vitro evaluation of biomarkers for cisplatin-induced kidney injury using HK2 cells treated with 10 μM UA-laden nanoparticles. Quantification of flow cytometry density plots of (a) TLR4, (b) NF-κB, and (c) IL-1β expression levels. Statistical analysis was carried out using two-way ANOVA. Significance levels were denoted as follows: **P < 0.01, ***P < 0.001, ****P < 0.0001.

At the 10 μM concentration, H-NPs (UA) were more effective than all other formulations in downregulating all three markers: TLR4, NF-κB, and IL-1β. It is note-worthy that L-NPs are less effective compared to P-NPs or H-NPs, probably due to multiple factors that include but are not limited to low loading, entrapment efficiency, a more excipient dump for similar UA concentrations, and slower release rates.

In summary, while all tested UA formulations at 10 μM concentration exhibited a capacity to reduce CIS-induced proinflammatory signaling in HK2 cells, the H-NPs (UA) formulation was particularly effective across all markers assessed. These findings underscore the potential of nanoparticle-mediated delivery of UA in mitigating the inflammatory response associated with CIS exposure in renal cells. Further investigations are warranted to elucidate the mechanistic differences between formulations and optimize therapeutic strategies for combating CIS-induced nephrotoxicity.

Conclusion

The development of a polymeric outer layer significantly improves the stability and structural integrity of H-NPs compared to L-NPs. Concurrently, the presence of a lipid-dense core enhances the affinity for lipophilic molecules, such as UA, more effectively than P-NPs. These combined features contribute to increased drug loading and entrapment efficiency for H-NPs, as supported by the results. CLSM structural analysis using fluorescent-labeled H-NPs confirms the formation of a polymer-rich outer layer surrounding a lipid-dense core nanoarchitecture, facilitating efficient GA superficial conjugation. Surface modification with GA promotes higher internalization of nanoparticles in intestinal and kidney cells compared to L-NPs, likely due to potential transferrin receptor-mediated uptake. Formulation safety is validated through FACS analysis, indicating no cytotoxic effects on these cells at the examined doses. Ultimately, H-NPs (UA) demonstrate the ability to downregulate the expression of proinflammatory markers in cisplatin-treated kidney cells compared to plain UA solution, L-NP (UA), and P-NPs (UA). This preliminary outcome suggests the potential of H-NPs (UA) as effective drug delivery systems for orally administering UA in the treatment of AKI, establishing a foundation for future in vivo investigations.