Targeted paclitaxel delivery in ovarian cancer via AP1-functionalized elastin-like polypeptide nanocarriers: development and characterization

Abstract

Paclitaxel has been a cornerstone of ovarian cancer chemotherapy for over two decades. However, its clinical application is constrained by poor solubility and non-specific delivery, resulting in systemic toxicity and inconsistent therapeutic outcomes. Nanotechnology-based drug delivery systems have emerged as a promising strategy to address these limitations. In this study, we employed elastin-like polypeptide (ELP) nanocarriers, precisely modified with the tumor-targeting AP1 peptide, to deliver paclitaxel in ovarian cancer. ELPs are biologically inspired, genetically engineered polymers that can form nano-sized structures with controlled physicochemical properties, facilitating passive tumor targeting. The integration of the AP1 peptide, which specifically binds to the IL-4 receptor overexpressed in numerous cancers, enables active targeting of these nanocarriers, complementing the passive delivery approach. This investigation focused on the synthesis and characterization of paclitaxel delivery vehicles based on modified (A60) and unmodified (E60) ELPs. Paclitaxel (PTX) was conjugated to ELPs via a thiol–maleimide Michael-addition strategy. Both ELP-PTX formulations formed stable, monodisperse micelles, with A60-PTX nanoparticles measuring 28 ± 2.8 nm and E60-PTX nanoparticles measuring 46.8 ± 6.6 nm, as determined by TEM. DLS analysis further confirmed the narrow size distribution, evidenced by a single, narrow peak in the size distribution profile, indicating near homogeneity of the micellar population. In vitro binding analysis in SKOV-3 and OVCAR-3 ovarian cancer cells demonstrated significantly enhanced targeting capability with A60, exhibiting ~ 8.6-fold and ~ 2.7-fold higher cell binding than E60, respectively. Consistently, A60-PTX demonstrated superior cytotoxicity, with ~ 2.6-fold and ~ 1.4-fold lower IC50 values than E60-PTX in SKOV-3 (47 nM vs. 120 nM) and OVCAR-3 (45 nM vs. 62 nM), respectively. The relevance of the active targeting was further validated in agarose-based 3D spheroid models of the two cell lines with A60-PTX demonstrating approximately ~ 3-fold (SKOV-3) and ~ 2.5-fold (OVCAR-3) higher cytotoxicity compared to E60-PTX. Overall, this study highlights the potential of AP1-functionalized ELP nanocarriers to enhance the precision and therapeutic efficacy of paclitaxel delivery, offering a promising strategy for targeted ovarian cancer therapy.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12885-026-15615-0.

Introduction

Ovarian cancer remains a prominent gynecologic malignancy, significantly contributing to female mortality rates worldwide [1]. For over two decades, the combination of paclitaxel and platinum-based agents, such as carboplatin, has been the preferred chemotherapeutic approach for the management of advanced ovarian cancer [2]. Paclitaxel, a well-established antineoplastic compound classified as an anti-microtubule agent, functions by inhibiting mitosis [3]. However, its conventional “naked” delivery method presents substantial challenges, including a lack of tumor specificity and associated severe undesired effects. Furthermore, the high hydrophobicity of paclitaxel, a trait common to numerous chemotherapeutics, limits its broader clinical application [4, 5]. Recent scientific endeavors have focused on developing innovative pharmacological strategies to address these limitations and enhance drug solubility and distribution. Notably, advancements in nanotechnology have led to the introduction of nanocarriers as promising vehicles for improved drug delivery. The encapsulation of small hydrophobic drugs in nanoscale particles enhance their pharmacokinetic and pharmacodynamic profiles. In addition, these nanostructures facilitate the optimal accumulation of therapeutic agents within target tissues. Considering these advancements, our research team opted to use a multifunctional genetically engineered nanoparticle to overcome the constraints associated with the administration of naked paclitaxel.

ELP exemplifies a biomimetic recombinamer [6–8]. The sequence of the polymer is derived from the hydrophobic domain of tropoelastin, an elastomeric monomer. Consequently, ELP exhibits the distinctive mechanical and self-assembly characteristics of its natural counterpart [9–11]. The genetic encoding of the polymer sequence enables precise modulation of parameters such as size, molecular weight, hydrophobicity, and architectural configuration, facilitating adaptation to diverse biomedical applications [12, 13]. The tailored approach to ELP synthesis, coupled with its biocompatibility, has proven particularly advantageous for drug delivery applications [14–18]. Notably, the versatile polymer has been extensively appraised as an enhanced permeability and retention (EPR)-based passive tumor targeting nanocarrier for the “selective” delivery of various therapeutic agents across multiple cancer types [15, 17, 19–24]. Previously, Sarangthem et al. leveraged the genetic encoding feature to embed “active targeting” elements within the polymer sequence, thus further enhancing its “targeting” capability [12, 25–27]. Specifically, the author periodically integrated multiple copies of CRKRLDRN, a peptide known as AP1, which demonstrates selective binding to the IL-4R. This strategy was informed by extensive literature on IL-4R overexpression in numerous cancer types [28–30]. Interestingly, meticulously engineered polymer sequences with varying molecular weights and architectures, all featuring multivalent presentation of the targeting peptide, demonstrate enhanced binding and uptake in vivo [12]. Thus, the multivalent display of AP1 peptide on the ELP scaffold yields a multifunctional polymer capable of both passive and active tumor targeting through the EPR effect and high-avidity interactions between the peptide-presenting polymer and IL-4R-expressing cancer cells.

In this study, we aim to utilize two of the previously defined macromolecules as delivery vehicles for paclitaxel. Specifically, a non-functionalized polymer (E60) and a ligand-functionalized polymer (A60) are strategically selected due to their similar molecular weights, with E60 serving as a non-targeting control. Furthermore, the drug is chemically modified to affix a thiol-reactive linker harboring a maleimide group to facilitate bioconjugation of the drug onto TCEP-reduced ELPs. Notably, the conjugated polymers exhibited self-assembly into nanoscale micelle-like architectures, demonstrating stability for over a period of three months. The formed micelles exhibited a low polydispersity index (PDI), addressing a major impediment associated with the use of chemically engineered delivery vehicles. Moreover, the conjugated drug continued to display cytotoxicity and metaphase-arrest properties associated with the free drug. Importantly, investigation of the cytotoxic ability of the drug-conjugated functionalized versus non-functionalized polymers in vitro and in spheroid models indicated the relevance of active targeting with AP1-functionalized ELP.

Materials and methods

ELP expression and purification

The A60 and E60 polymers were cloned in pET-24b expression plasmid and were transformed into chemically competent BL21 (DE3) E. coli cells, as previously described [12]. An initial culture was prepared using the transformed colonies in 15 mL of Terrific Broth with 100 µg/mL ampicillin and incubated overnight at 37 °C and 180 rpm. The starter culture was then used to seed a larger 750 mL batch of Terrific Broth containing antibiotics. The secondary cultures were grown at 37 °C and 180 rpm until reaching an OD600 of 0.8-1. Protein expression was then induced with 1 mM IPTG, and incubation was continued for 4 h under the same conditions. The cultures were subsequently harvested, and the resulting pellet was resuspended in PBS. Cell lysis was performed by sonicating the resuspended cells at 4 °C, followed by protein purification using the inverse transition cycling (ITC) method. Three rounds of ITC were conducted to ensure ELP purity, which was further verified through SDS-PAGE analysis. The concentration of the purified polymers was determined using UV-Vis spectroscopy, with an extinction coefficient of 5690 M − 1 cm − 1.

Synthesis of the paclitaxel analog compound X

The activation of paclitaxel (PTX) for biopolymer conjugation was carried out using the maleimide-based linker 6-maleimidocaproic acid (EMCA), following the method previously described by Zhang et al. [31]. Briefly, 30 mg of EMCA, 67.6 mg of dicyclohexylcarbodiimide (DCC), and 5 mg of 4-dimethylaminopyridine (DMAP) were dissolved in 6 mL of anhydrous dichloromethane (DCM). This mixture was stirred for 1 h at 0 °C under nitrogen. Subsequently, 70 mg of PTX, dissolved in 9 mL of anhydrous DCM, was added dropwise to the mixture. The reaction continued overnight in the dark at 25 °C under nitrogen. After working up the reaction mixture, its completion was evaluated by Thin Layer Chromatography. Mass Spectrometry (MS) was employed to confirm the successful synthesis of the desired PTX analog. Following confirmation, the desired PTX analog was isolated and purified through silica column chromatography using a 1.5:1 ratio of ethylacetate to hexane eluent. The chemical identity of the desired fraction was verified using ¹H NMR and HRMS techniques.

Conjugation of compound X with ELPs

To achieve stable conjugation of Compound X with ELP, the classical Michael-addition bioconjugation method was utilized. Initially, ELPs A60 and E60 underwent reduction with TCEP at neutral pH, using a 5-fold excess relative to thiol. This reduction process was conducted at room temperature for 1 h. Subsequently, excess TCEP was eliminated in one round of ITC. The resulting reduced protein pellet was resuspended in 1 M PBS solution. Compound X, dissolved in DMSO, was slowly and intermittently introduced into this solution. The mixture was then stirred overnight at 4 °C in the dark. Subsequently, any unreacted Compound X was removed by centrifuging at 13,000 r.p.m. at 4 °C for 10 min. Further purification was achieved by centrifuging the solution in Amicon Ultra-0.5 Centrifugal Filter Units (MWCO: 10 kDa, Millipore) at 4 °C.

MALDI TOF/MS analysis

The molecular weights of the conjugated ELP nanopolymers and free polypeptides were determined using an Autoflex Max TOF/TOF MALDI (Bruker). The spectra were obtained after calibration using standards.

Dynamic light scattering (DLS) analysis and zeta potential measurement

DLS was performed to determine the hydrodynamic radius (Rh) of the conjugated ELPs at a concentration of 25 µM at 37 °C using a SpectroSize 300 instrument (Xtal Concepts, Hamburg, Germany). Zeta potential measurements were performed to assess the surface charge of the conjugated ELPs using a Zetasizer instrument (Malvern Instruments, Worcestershire, UK).

Transmission electron microscopy (TEM)

To confirm the micelle formation following drug conjugation, Tecnai HR-TEM at the Sophisticated Analytical Instrumentation Facility (SAIF) of AIIMS, New Delhi was employed. ELP-PTX nanosuspensions in PBS were placed on carbon-coated copper grids and allowed to air dry for 5 min. The samples were then negatively stained with uranyl acetate before TEM examination.

Drug release assay

The release of paclitaxel from nanoformulations (E60-PTX and A60-PTX) was evaluated utilizing the dialysis diffusion method. Briefly, 2 ml of the nanoparticle formulation was introduced into a dialysis membrane with a molecular weight cut-off (MWCO) of 12,000–14,000 Da. The suspension was dialyzed against 100 ml of phosphate-buffered saline (PBS, pH 7.4) at 37 °C under gentle agitation at 60 rpm. At specific time intervals, 2 ml of the external medium was extracted for analysis and replaced with fresh PBS to maintain sink conditions. The drug concentration was quantified using UV-Vis spectrophotometry at 226 nm.

Cell culture

The SKOV-3 and OVCAR-3 human ovarian cancer cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in RPMI (Rosewell Park Memorial Institute, Thermo Fisher Scientific, Watham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin-streptomycin solution. Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. Paclitaxel (99.5% purity) was purchased from LC Labs (Woburn, MA 01801).

In vitro cell binding assay

The selectivity of A60 polymer for IL-4R, in comparison to its non-functionalized counterpart E60, was investigated using ovarian cancer cell lines, SKOV-3 and OVCAR-3, both of which are reported to highly express IL-4R. Briefly, 4 × 10⁵ cells were incubated with 500 nM Alexa-488-labeled ELPs in PBS + 1% BSA at 37 °C for 1 h. After washing the cells twice with PBS, flow cytometry analysis was performed using a flow cytometer (BD FACScan Biosciences, Mountain View, CA, USA). For each sample, 10,000 events were collected.

Cell proliferation assay

The Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan) was employed to assess cell viability. Briefly, ovarian cancer cell lines (SKOV-3 and OVCAR-3) were seeded into 96-well plates at a concentration of 10 × 10³ cells per well. After a 24 h incubation period, the cells were exposed to various concentrations of PTX/A60-PTX/E60-PTX for 48 h. Subsequently, 10 µL of CCK-8 reagent was added to each well, followed by an additional 1 h incubation. A BioTek Synergy HT Multi-Mode microplate reader (Winooski, VT, USA) was used to measure the absorbance at 450 nm, providing a quantitative measure of cell viability.

Apoptosis and cell cycle analysis by flow cytometry

Flow cytometry based Annexin V-Elab Fluor^® 488/PI Apoptosis Kit (Elabscience) and propidium iodide (PI) staining were used to assess apoptosis and cell cycle distribution. 2 × 10⁵ cells were plated in each well of a six-well plate. After 24 h incubation at 37 °C in a 5% CO₂ environment, the cells were treated with an appropriate dosage of A60/E60-PTX, followed by an additional 48 h of incubation under identical conditions. The treated cells were harvested distinctly for apoptosis and cell cycle distribution analysis as per the manufacturer’s instructions.

Fluorescence signals from both the assays were detected using flow cytometer. The resulting data was examined using FlowJo software v10.0.7 (Ashland, OR, USA).

Spheroid culture

Agarose-coated 96-well plates were used to generate 3D spheroids. A solution of 1.5% (w/v) agarose in sterilized PBS was heated to ensure complete dissolution, yielding a homogenous solution. The solution was then distributed into each well of a 96-well plate and allowed to gelate at ambient temperature. Subsequently, 10,000 cells of the respective cell lines were introduced into the coated wells. The plates underwent incubation under standard conditions (37 °C, 5% CO₂, 95% humidity) for a minimum of two days. An inverted light microscope was used to observe spheroid formation. On the third day, the formed spheroids were subjected to treatment with free drug and the corresponding nanoformulations. Following a 48 h treatment period, the effects on the spheroid model were assessed using a Viability/Cytotoxicity Assay Kit (Biotium, San Francisco, USA) featuring calcein AM and EthD-III dyes, in accordance with the manufacturer’s guidelines. Visualization of the stained spheroids was accomplished using a Leica DM6 Microscope (Wetzlar, Germany).

Statistical analysis

All statistical analyses were performed using GraphPad Prism software. Data are presented as mean ± standard deviation (SD). Student’s t-test was used for pairwise comparison of percentage cell viability with E60-PTX vs. A60-PTX at specific doses, while one-way ANOVA followed by Tukey’s post-hoc test was applied for multiple group comparisons where appropriate. A P-value of less than 0.05 was considered statistically significant.

Results

1. Synthesis and functional characterization of PTX analog compund X

In this study, we used the conventional thiol-maleimide Michael-addition method of bioconjugation of drugs to macromolecules [32]. The chemical process, involving a maleimide acceptor and a thiolate donor, leverages strategically positioned cysteine residues within protein polymers to control drug conjugation. The conjugation reaction was rationally strategized into two key steps. The initial phase involved activating the antineoplastic agent using EMCA, a bifunctional linker featuring maleimide and carboxylic acid groups at the opposite termini. The carboxylic moiety facilitates activation through ester bond formation with the drug’s hydroxyl group (Fig. 1A). Paclitaxel, a complex diterpene, with a rigid central core composed of fused rings and a comparatively flexible C-13 side chain. This side chain is integral to tubulin binding, with the 2′-OH group being indispensable for activity, establishing a stable hydrogen bond with tubulin, the natural substrate [33]. Interestingly, this fundamental structural element has been widely employed in synthesizing drug analogs [34]. Our research utilized this critical residue for drug activation employing a maleimide-based linker, following the methodology described by Zhang et al. [31].

Fig. 1.

Synthesis and functional characterization of PTX analog, compound X (A) Steglich Esterification reaction between sterically demanding 2’OH of the paclitaxel and carboxyl group of the linker, EMCA. B SKOV-3 cells were treated with varying concentrations of native drug, PTX or the drug analog, Compound X for 48 h. Cell viability was measured with CCK-8 assay. C Cell cycle distribution of SKOV-3 cells after 24 h exposure to increasing concentrations of Compound X was assessed using Flow cytometry

The initial successful synthesis of the PTX analog referred as Compound X was confirmed through HRMS analysis (Fig. S1), and further verified using ¹H NMR spectroscopy (Figure S2). Furthermore, the cytotoxic properties and metaphase-arrest capabilities of the activated drug analog were substantiated through CCK-8 assay (Fig. 1B) and FACS-based cell cycle analysis (Fig. 1C, Fig. S3). The activated drug inherited the native cytotoxic ability and metaphase-arrest property. The reduced cytotoxicity could be attributed to rate limiting esterase-triggered hydrolysis of the drug analog to release the free drug.

• 2.Bioconjugation of PTX to the ELP polymer

The subsequent phase in the bioconjugation process involved the incorporation of a cysteine-harboring sequence into the previously described ELP polymers, A60 and E60. A nucleotide sequence with six cysteine codons (Fig. S4), each separated by two amino acids, was introduced to reduce steric hindrance between neighboring drug analogs. The in-frame insertion of the polynucleotide was verified using Sanger sequencing. Subsequently, the conventional Michael-addition reaction was employed to attach the drug to the polymer template (Fig. 2A). A calculated molar ratio of Compound X to ELP was employed to optimize conjugation efficiency. The initial assessment of drug conjugation was performed using SDS-PAGE analysis, where the observed shift in polymer mobility indicated successful conjugation (Fig. 2B). Further, the drug-to-polymer conjugation ratio was determined via MALDI-TOF analysis (Fig. 2C). The mass difference between the conjugate and the parent polymer indicated the attachment of 4 and 5 PTX molecules per E60 and A60 polymer, respectively.

Fig. 2.

Bioconjugation of paclitaxel to the ELP polymer (A) A schematic diagram illustrating the bioconjugation of maleimide activated PTX to TCEP reduced thiol residues at the C-terminus of the ELP polymers, as adapted from reference [12]. B Assessment of bioconjugation of PTX to the ELP polymers by SDS-PAGE analysis. The electrophoretic mobility shift from the association of the drug was visualised by Coomassie Blue staining. C MALDI-TOF analysis of the native and drug conjugated ELP polymers

• 3.Characterization of PTX-loaded ELP nanopolymers

Genetically engineered ELPs offer a unique capability to create diverse nanoscale structures. The nanoscale dimensions of the polymeric carrier facilitate targeted tumor delivery through the EPR effect. McDaniel et al. demonstrated that attaching maleimide derivatives with sufficient hydrophobicity (Log(D) exceeding 1.5) to water-soluble ELPs (Tt > > 37 °C) triggers the spontaneous assembly of nanoscale micellar architectures from soluble ELP monomers [35]. Building on this finding, we anticipated that the stable binding of highly hydrophobic paclitaxel, with a Log(D) of 4.95, would induce the self-assembly of ELPs, A60 and E60 into nanoscale micellar formations. DLS and TEM analyses were conducted to characterize the physicochemical properties of the conjugated ELP polymers. TEM analysis showed nearly monodisperse spherical micellar-like structures for both drug-conjugated ELPs (Fig. 3A). TEM measurements indicated that A60-PTX nanoparticles were 28 ± 2.8 nm in size, while E60-PTX nanoparticles were 46.8 ± 6.6 nm. Additionally, DLS analysis verified the narrow size distribution of the conjugated nanoscale ELP micelles (Fig. 3B). The hydrodynamic radii from DLS analysis of the ELP-PTX polymers were approximately Rh of 92.82 nm for A60-PTX and Rh of 72.65 nm for E60-PTX. The apparent discrepancy between the particle sizes observed via TEM and those obtained from DLS is well documented in literature and can be attributed to differences in the measurement principles of these techniques. TEM provides a dry-state measurement of the electron-dense core structure under high vacuum. In contrast, DLS measures the hydrodynamic diameter of particles in solution, which includes not only the dense core but also the surrounding hydration shell.

Fig. 3.

Characterization of PTX-loaded ELP nanopolymers (A) Assessment of the morphology and size of the ELP-PTX nanopolymers using TEM. B Assessment of the hydrodynamic radii of the ELP-PTX nanopolymers using DLS

Notably, conjugation driven spontaneously assembled nanopolymers exhibited stability for an extended period of three months maintaining similar size distribution characteristics as confirmed by DLS (Fig. S5).

Further, to assess the surface charge characteristics of the conjugated ELP micelles, zeta potential measurements were performed on E60-PTX and A60-PTX nanoparticles. E60-PTX exhibited a zeta potential of − 2.48 mV, whereas A60-PTX had a slightly positive value of + 3.69 mV. Although uncommon, near-neutral zeta potentials have previously been observed in ELP-based nanoformulations, including those loaded with rapamycin and hybrid systems combining ELPs with liposomes for docetaxel delivery [22, 36]. Importantly, nanoparticles with near-neutral surface charges are often considered beneficial in drug delivery. Strongly negative particles (zeta potential < − 10 mV) are more likely to be rapidly cleared by the reticuloendothelial system (RES). On the other hand, particles with high positive charges (zeta potential > + 10 mV) can induce serum protein aggregation. In contrast, nanoparticles with zeta potentials close to neutral (within ± 10 mV) are generally associated with extended circulation time and reduced RES clearance [37, 38].

The functional stability and release characteristics of the paclitaxel-conjugated micelles were further evaluated using a dialysis-based method. Both A60-PTX and E60-PTX formulations demonstrated a controlled, biphasic release profile (Fig. S6). An initial release of approximately 30–40% was observed within the first 20 h, likely corresponding to the diffusion of non-covalently associated paclitaxel from the micellar core. Beyond this point, the release plateaued, with minimal further drug release detected up to 48 h, indicating that the remaining paclitaxel was covalently attached and released slowly, likely through hydrolytic cleavage of the linker. This sustained release profile reflects the stability of the nanoparticle formulation and suggests potential for prolonged therapeutic effect with reduced dosing frequency.

• 4.In vitro cell binding of ELP polymers

The current investigation builds upon the selective targeting capabilities of AP1 peptides attached to the ELP biopolymer. Our prior research demonstrated the exceptional targeting efficacy of the A60 polymer in a breast cancer model [12]. Interestingly, ovarian cancer tissues exhibit elevated IL-4R expression compared to normal ovarian tissues ( Kioi et al.), suggesting a potential extension of the polymer’s selectivity to this malignancy. IL-4R expression has also been detected in ovarian cancer cell lines, such as SKOV-3 and OVCAR-3 [29], rendering these cell lines suitable for evaluating the efficacy of our polymers in targeting paclitaxel actively to ovarian cancer cells. Differential binding of the ligand-functionalized polymer with multivalent display (A60) versus the non-functionalized polymer (E60) was evaluated in the ovarian cancer cell lines using flow cytometry (Fig. 4).

Fig. 4.

In vitro cell binding of the ELP polymers. Percentage cell binding of the ELP polymers in (A) SKOV-3 and (B) OVCAR-3. The cells were incubated with 500 nM Alexa-488 labeled ELPs for 1 h at 37 °C. Cell binding of the polymers was determined using flow cytometry. Histograms are representative of three independent experiments. *p < 0.05, ***p < 0.001 (one-way ANOVA)

The analysis showed that the A60 polymer had significantly higher cell binding capability than E60 in both the cell lines. In particular, E60 showed cell binding activities of 10.73 ± 2.5%, whereas the targeting polymer displayed significantly higher binding capacities of 92.2 ± 4% (p = 0.0002) in SKOV-3. Similarly, in OVCAR-3, E60 demonstrated 30.16 ± 3% cell binding compared to 81.67 ± 7.5% for A60 (p = 0.0006). The distinct specificity of the A60 polymer in the two cell lines could be attributed to differential IL-4R expression in these cell lines.

• 5.In vitro cytotoxicity of PTX-loaded ELP nanopolymers in ovarian cancer

The antiproliferative activity of the drug-loaded nanopolymers was evaluated using CCK-8 assay. Initially, the cytocompatibility of the native ELP biopolymers (A60/E60) was assessed in the cells. As illustrated in Fig. S7, cells treated with the biopolymers exhibited high cell viability and minimal toxicity at concentrations fold-times the anticipated IC50 of the drug after 24 h and 48 h of incubation. Consequently, biopolymers alone serve as suitable vehicles with favorable safety. Subsequently, the proliferation inhibition effect of the nanopolymers was examined across a concentration range (drug-equivalent) in comparison to the free drug. The corresponding cytotoxicity profiles are presented in Fig. 5A and C.

Fig. 5.

In vitro cytotoxicity of PTX-loaded ELP nanopolymers in ovarian cancer cells. A SKOV-3 and (C) OVCAR-3 cells were treated with varying concentrations of PTX/ELP-PTX (3 nM to 250 nM) for 48 h. Cell viability was measured with CCK-8 assay. Cell viability in (B) SKOV-3 and (D) OVCAR-3 at selected doses of E60/A60-PTX following 48 h exposure. *p < 0.05, **p < 0.01 compared with E60-PTX

Notably, both biopolymers loaded with the drug exhibited dose-dependent inhibition of proliferation. In particular, the ligand-functionalized polymer exhibited markedly enhanced cytotoxicity in both the cell lines (Table 1), with IC50 values of 47 nM in SKOV-3 and 45 nM in OVCAR-3. These values represent approximately 2.6-fold and 1.4-fold reductions, respectively, compared to the non-functionalized counterpart (E60-PTX, 120 nM and 62 nM, respectively), underscoring the efficacy of the targeted treatment approach. The enhanced cytotoxicity of the targeting polymer is further exemplified in the Fig. 5B and D by comparing the cytotoxicity of the PTX-loaded polymers at specific doses. The discrete window of selective targeting associated with differential cytotoxicity in the two cell lines corroborate the distinctive polymer specificity of the two cell lines (Fig. 4).

Table 1.

IC50 values of PTX/ELP-PTX in SKOV-3 and OVCAR-3 cells

	IC50 values (nM)
Drug	SKOV-3	OVCAR-3
PTX	20	6
E60-PTX	120	62
A60-PTX	47	45

• 6.Effects of PTX-loaded polymers on drug-associated cell cycle arrest and apoptosis

The direct interaction of the anticancer agent paclitaxel with the beta subunit of tubulin and subsequent stabilization of microtubule dynamics is known to impede cellular progression through the G2/M phase of the cell cycle, ultimately leading to apoptosis. We investigated the efficacy of the ELP-conjugated drug in inducing cell cycle arrest and programmed cell death. To achieve this, cells were subjected to PI and Annexin V/PI staining after 48 h exposure to appropriate drug concentrations. In cell cycle experiments, concentrations were carefully selected to ensure substantial cell viability. The distribution of cells across various cell cycle phases in SKOV-3 is illustrated in the histogram (Fig. 6A).

Fig. 6.

Effect of PTX-loaded polymers on drug-associated cell cycle arrest (A-C) Flow cytometry-based cell cycle analysis of SKOV-3 cells after exposure to E60-PTX/A60-PTX (15 nM) for 48 h. **p < 0.01 (one-way ANOVA)

Cell cycle analysis revealed distinct distribution patterns among the treatment groups (Table 2). Both control and E60-PTX treated cells displayed similar profiles, with the majority of cells residing in the G0-G1 phase and relatively low percentages in the G2-M phase. In contrast, A60-PTX treatment induced a notable shift, marked by a decrease in G0-G1 phase cells and a concurrent increase in the G2-M phase, reflecting mitotic arrest, a characteristic of native paclitaxel (Fig. 6B).

Table 2.

Percentage of cells in each cell cycle phase (Mean ± SD)

Treatment	G0-G1 (%)	S Phase (%)	G2-M (%)
Control	73.97 ± 4.20	11.30 ± 1.04	16.00 ± 4.00
E60-PTX	72.17 ± 2.00	12.33 ± 2.52	15.50 ± 0.50
A60-PTX	54.73 ± 2.60	16.47 ± 1.33	28.80 ± 1.31

Importantly, the increase in G2-M population following A60-PTX treatment was statistically significant compared to both control (p = 0.0017) and E60-PTX groups (p = 0.0014) (Fig. 6C). This improved activity at the tested dose likely results from more efficient cellular internalization of the ligand-targeted A60-PTX nanoparticles, leading to increased intracellular paclitaxel availability and consequent biological activity.

Moreover, the apoptosis analysis further emphasized the significance of targeted PTX delivery with A60 biopolymer. As depicted in Fig. 7A, B, treatment with 50 nM PTX equivalent A60-PTX induced apoptosis in 35% of SKOV-3 cells and 33.1% of OVCAR-3 cells, compared to 17.5% and 11% in the corresponding E60-PTX–treated groups. This represents a twofold increase in SKOV-3 (p = 0.0014) and a threefold increase in OVCAR-3 cells (p = 0.0022), confirming the enhanced cytotoxicity conferred by IL-4R–targeted drug delivery.

Fig. 7.

PTX-loaded polymers induced apoptosis in ovarian cancer cells. Annexin V/PI dual staining method for apoptosis analysis of (A) SKOV-3 cells and (B) OVCAR-3 cells after exposure to E60-PTX/A60-PTX (50 nM) for 48 h. **p < 0.01, ***p < 0.001 (one-way ANOVA)

Effect of PTX-loaded polymers in 3D spheroid cultures

After demonstrating the cytotoxicity of ELP-PTX polymers in monolayer cultures, we assessed the biopolymer-facilitated drug delivery in a 3D model. This approach was based on the understanding that in vitro 3D cancer spheroids function as miniature versions reflecting in vivo tissue physiology and complexity. These cellular clusters notably display gene expression patterns that more accurately mirror the biology of their corresponding tissues, thus providing a better platform for evaluating drug effectiveness [39]. We hypothesized that the enhanced targeting capability of the modified ELP polymer would be more evident in multicellular 3D models.

Various methods exist for creating spheroids, all of which are aimed at preventing cell attachment to the culture vessel surface, thereby enhancing interactions between neighboring cells and the extracellular matrix. We used a straightforward liquid-overlay technique to coat the wells with agarose solution before adding the cell suspension. This non-adhesive coating promotes cell-to-cell contact as cells gather on the concave bottoms. By day 3 of seeding, we successfully cultivated one spheroid per well using this method. Once the compact spheroids formed, we administered PTX and the ELP-PTX polymers. We carefully selected doses that were fold times the concentrations used in monolayer cultures, considering the known increased resistance to drugs in more complex 3D environments. We assessed the cytotoxicity of these drugs using cell-permeant calcein/AM and non-permeant ethidium homodimer-3. In both the cell lines, A60-PTX exhibited notably superior performance, as evidenced by a marked increase in red fluorescence resulting from EthD-III binding to nucleic acids in dead cells with compromised membrane permeability, when compared to E60-PTX and PTX treatments (Fig. 8A-D). It is important to note that in the intricate 3D cellular structure, the passive diffusion of free drugs is more restricted than in monolayer cultures, leading to reduced cytotoxicity of the free drug. Moreover, the unique receptor expression in the 3D cell organization, which more closely mirrors in vivo conditions, underscores the enhanced importance of the ligand-functionalized polymer in drug delivery. This highlights the significance of these models in drug screening.

Fig. 8.

PTX-loaded polymers induced cell death in 3D spheroid ovarian cancer cells. A, C SKOV-3 and (B, D OVCAR-3 cells were originally plated at 10,000 cells per well in an agarose coated 96-well plate. On day 3, spheroids formed were treated with test drugs, PTX and PTX-loaded polymers. Following 48 h of treatment the effect of the drugs was assessed using Calcein-AM and EthD-III staining. **p < 0.01, ***p < 0.001 (one-way ANOVA)

Discussion

Paclitaxel is a frontline chemotherapeutic for various solid tumors, including ovarian cancer. However, its clinical utility is limited by poor solubility, suboptimal pharmacokinetics, nonspecific distribution, and dose-limiting toxicities. To overcome these barriers, several nanoparticle-based paclitaxel formulations have been developed. Among these, albumin-bound paclitaxel (nab-paclitaxel, Abraxane) was the first to gain FDA approval, followed by liposomal paclitaxel (Lipusu) and polymeric micelles (Genexol-PM) [40–42]. However, these platforms continue to grapple with concerns over long-term toxicity, unclear survival advantages, high costs, and manufacturing complexity. Consequently, there remains an urgent demand for alternative paclitaxel delivery systems that can achieve targeted, efficient, and scalable clinical translation.

In this regard, elastin-like polypeptides represent a promising and versatile drug delivery platform, combining excellent biodegradability, biocompatibility, and genetic encodability. The absence of post-translational modifications enables their efficient production in bacterial systems, while their reversible phase transition behavior allows simple, chromatography-free purification via inverse transition cycling [16]. This streamlined, cost-effective, high-yield production addresses a critical bottleneck faced by many synthetic nanoformulations, high cost of production. Moreover, the inherent genetic tunability of ELPs allows seamless incorporation of tumor-specific targeting ligands, advancing beyond many existing paclitaxel nanocarriers that depend primarily on passive accumulation through the enhanced permeability and retention effect for selective drug delivery. For instance, earlier studies have reported that the multivalent display of the NGR peptide on ELP block copolymer micelles substantially boosts vascular retention and tumor-specific accumulation by interacting with the tumor vasculature marker CD13, in contrast to normal tissue [43].

Targeted delivery can also be directed against overexpressed cancer cell receptors. IL-4R is a notable target, being highly expressed in various solid tumors, including ovarian cancer [28–30]. Building upon this rationale, Sarangthem et al. previously reported decoration of ELPs with the IL-4R-targeting peptide AP1 significantly enhances their tumor-targeting specificity and intratumoral retention, leading to improved tumor suppression in vivo [12, 26, 27]. The current study expands this strategy to deliver paclitaxel in ovarian cancer.

The previously characterized A60 polymer alongside its ligand-deficient counterpart E60, was evaluated for IL-4R mediated uptake in ovarian cancer cell lines, following its known differential binding in breast cancer model [12]. In line with earlier observations in MDA-MB-231 and 4T1 cells, A60 demonstrated significantly higher binding than E60 (p < 0.001). This finding emphasizes on the relevance of AP1-based targeting in IL-4R positive ovarian cancer. Further, a clinically validated thiol–maleimide click chemistry, widely employed in FDA-approved antibody–drug conjugates [44], enabled efficient and site-specific conjugation of paclitaxel, yielding stable micellar nanostructures with extended shelf-life. Consistent with earlier ELP–PTX formulations reported by the Chilkoti group [15, 24], both A60-PTX and E60-PTX assembled into sub-100 nm, near-monodisperse micelles. In 2D culture experiments, functional assessments underscored the biological relevance of the targeted delivery system. The AP1-functionalized A60-PTX polymer exhibited significantly increased cytotoxicity compared to the non-targeted E60-PTX in both SKOV-3 and OVCAR-3 cell lines, with IC50 values of 47 nM and 45 nM, respectively. These values indicate improvements of 2.6-fold and 1.4-fold over E60-PTX. The results were corroborated by apoptosis assays, which indicated 2-fold and 3-fold increase in apoptosis in SKOV-3 and OVCAR-3 cells, respectively. For the first time, these results affirm the benefit of IL-4R targeted delivery of paclitaxel and emphasises the adaptability of this delivery platform for chemotherapeutic applications. To further validate the platform, the enhanced efficacy conferred by AP1 was assessed in 3D spheroid models. Consistent with the 2D findings, A60-PTX exhibited superior performance compared to the non-targeted E60-PTX, demonstrating a 3-fold (p = 0.0007) and 2.5-fold (p = 0.0009) increase in efficacy for SKOV-3 and OVCAR-3 spheroids, respectively. Notably, in the 3D context, A60-PTX also outperformed free paclitaxel, highlighting its improved penetration. These results underscore the advantage of active IL-4R targeting in overcoming the diffusion limitations typically associated with native drug formulations.

Building upon the encouraging results in 2D and 3D models, future in vivo studies will be essential to further validate the therapeutic potential of IL-4R targeted ELP-PTX conjugates in ovarian cancer. These investigations will allow assessment of pharmacokinetics, biodistribution, tumor accumulation, and antitumor efficacy in a more physiologically relevant context. Evaluating A60-PTX in established ovarian tumor xenografts will be particularly informative in confirming the advantages of active targeting over passive delivery systems. Beyond tumor-specific delivery, targeting IL-4R may present an opportunity to modulate the tumor microenvironment. Recent investigation show that both genetic and pharmacological inhibition of IL-4Rα reduce CD206 expression in tumor associated macrophages (TAMs), shifting them away from the pro-tumor M2-like phenotype [45]. Consequently, the use of A60-PTX polymer could facilitate a dual-action therapeutic strategy, simultaneously target cancer cells and alter the immune milieu to enhance therapeutic efficacy.

Conclusion

In recent times, ELP-based polymers have emerged as highly promising platforms for drug delivery due to their versatility. These polymers can be genetically encoded, a feature that has been extensively exploited to design a wide array of nanoscale structures that enhance selective tumor targeting through the EPR effect. Despite this progress, there is still significant potential in exploring the precise modification of these polymers to achieve active targeting, which could capitalize on the expanding knowledge of molecular markers in cancer.

In this research, we investigated for the first time the application of ELPs to deliver paclitaxel by specifically targeting the unique molecular marker of IL-4R overexpression found in cancer cells. Towards this, we utilized our previously characterized ELP-based polymers, A60 and E60. The AP1–modified A60 polymer, engineered with IL-4R specificity, demonstrated significantly enhanced binding affinity to ovarian cancer cells compared to the non-functionalized E60 control. Michael-addition-based bioconjugation technique was employed for the synthesis of polymer-payload (ELP-PTX) conjugates, which demonstrated efficient payload loading and prolonged stability. As reported earlier, both polymers displayed attachment-triggered spontaneous assembly into monodisperse nano-sized micellar architectures. Cytotoxicity assays revealed an increase in anti-cancer activity for A60-PTX nanopolymers relative to E60-PTX, underscoring the therapeutic advantage conferred by active targeting. Additionally, the functionalized nanopolymers demonstrated superior apoptosis induction, further confirming their selective tumor-targeting capability. The therapeutic benefit of active delivery was particularly pronounced in 3D spheroid models, likely reflecting the complex gene expression differences between 2D monolayers and more physiologically relevant 3D tumor environments.

Although our findings are preliminary and require further validation in mouse models, the innovative polymer design and the robustness of the resulting nanopolymers open up numerous possibilities for ELP-based delivery systems. These systems have the potential to enhance targeted delivery by drawing on the extensive body of research on the genetic and molecular profiles of cancer cells.

Authors’ contributions

R.G, V.S and T.D.S contributed to the study’s conception and design. S.A, R.A, P.S, J.B, help in the synthesis of biopolymer and drug conjugates. R. G. wrote the first draft of the manuscript, and T.D.S proof read the final draft of the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by Indian Council of Medical Research (ICMR), No. 34/17/2019-TF/Nano/BMS, (IIRP-2023-0914) and Anusandhan National Research Foundation (ANRF), (CRG/2023/001373).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

No human samples and animal studies involved. All the cell lines experiments were performed as per applicable institutional guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.