Intracellular Delivery of a p21-Derived Cell Cycle Inhibitory Peptide Using Elastin-like Polypeptides Suppresses Glioblastoma Cell Proliferation

Abstract

Glioblastoma, with a 5-year survival rate of just under 7.0%, is the most common form of brain cancer in adults. In this study, we evaluated the antiproliferative activity of the biopolymer p21-ELP1-Bac, a p21-derived peptide delivered via an elastin-like polypeptide (ELP1) carrier and a cell-penetrating peptide (CPP), across three glioblastoma cell lines: U87, GBM43, and GBM6. We assessed proliferation, cell cycle progression, and apoptosis to determine whether ELP-mediated intracellular delivery of p21-ELP1-Bac suppresses glioblastoma growth through cytostatic mechanisms rather than inducing apoptosis. Treatment with the modified protein effectively inhibited proliferation across all three lines, with U87 cells showing the greatest sensitivity and GBM6 cells demonstrating the greatest drug tolerance. Although apoptotic responses were generally low, they appeared more pronounced in GBM6 cells. Confocal microscopy revealed sustained cellular uptake and signal observed in both the cytoplasm and in proximity to the nucleus in all cell lines. Collectively, these findings indicate that p21-ELP1-Bac is efficiently internalized and capable of modulating proliferation across all three glioblastoma cell lines, supporting its further evaluation as a cytostatic delivery platform.

1. Introduction

Glioblastoma (GBM), historically referred to as glioblastoma multiforme, is classified as a grade IV malignancy under the World Health Organization (WHO) criteria [1]. A key feature of GBM is disruption of tumor suppressor pathways, particularly p53 and its downstream effector p21, leading to dysregulated cell-cycle progression, uncontrolled cellular proliferation, and apoptotic evasion [2]. As a cyclin-dependent kinase (CDK) inhibitor, p21 regulates cell-cycle arrest at the G1/S and G2/M checkpoints and participates in DNA damage responses, apoptosis, and senescence-associated signaling. Restoration or enforced expression of p21 has been shown to suppress tumor growth and enhance sensitivity to chemotherapeutic agents in multiple cancer models, supporting its relevance as a therapeutic target in malignancies characterized by aberrant cell-cycle control.

Despite its therapeutic potential, the clinical application of p21-derived peptides is limited by poor intracellular delivery, rapid proteolytic degradation, and unfavorable pharmacokinetic properties [3]. These limitations are particularly pronounced in brain tumors, where the blood–brain barrier (BBB) severely restricts delivery of exogenous proteins and peptides to tumor tissue [4]. Therefore, effective strategies that enhance intracellular uptake and stability are required to enable translation of p21-based therapies for glioblastoma.

Engineered delivery platforms, including cell-penetrating peptides (CPPs) and elastin-like polypeptides (ELPs), have been developed to overcome these barriers. CPPs facilitate translocation across cellular membranes, enabling cytosolic and nuclear-associated delivery of therapeutic cargo. ELPs are biocompatible polymers composed of repetitive pentapeptide motifs that confer resistance to proteolytic degradation and prolonged circulation time. When used as carrier scaffolds, ELPs can be engineered to incorporate CPP domains and targeting sequences, thereby improving intracellular delivery, retention, and tumor-associated accumulation of peptide therapeutics.

Our laboratory has previously demonstrated the efficacy of ELP-mediated delivery of p21-derived peptides in prostate and ovarian cancer models. In androgen-independent prostate cancer, ELP-based delivery of a C-terminal p21 mimetic enhanced intracellular stability and uptake, resulting in CDK inhibition, cell-cycle arrest, and reduced tumor cell proliferation, particularly in combination with chemotherapeutic agents [5]. Similarly, in ovarian cancer models, p21–ELP-based delivery platforms exhibited improved bioavailability, increased nuclear adjacent localization, and greater antiproliferative activity compared to unconjugated peptides, inducing cell-cycle arrest and apoptotic signaling while overcoming intrinsic delivery limitations [6]. These findings established ELPs as effective carriers for intracellular delivery of p21-derived therapeutics.

In the present study, this delivery platform is extended to glioblastoma by evaluating the effects of ELP-mediated p21 delivery in three GBM cell lines: GBM43, U87, and GBM6. These models represent distinct glioblastoma phenotypes, including patient-derived, therapy-resistant tumors and a highly proliferative established cell line. Cell proliferation, cell-cycle distribution, apoptotic response, and subcellular uptake and localization of the p21–ELP therapeutic were assessed to determine delivery-dependent effects on glioblastoma cell growth. Building on prior work in prostate and ovarian cancer models [5,6], this study examines the responsiveness of heterogeneous GBM subtypes to intracellular p21 delivery and supports the broader applicability of this delivery strategy in aggressive brain cancers.

2. Results

2.1. Design of ELP-Based Drug Delivery Macromolecule

The p21-ELP1-Bac carrier system consists of a p21-derived inhibitory peptide fused to an elastin-like polypeptide carrier and a cell-penetrating peptide, enabling intracellular delivery of p21 in glioblastoma cells. This macromolecular drug delivery system is engineered for efficient intracellular delivery of p21 [7]. The delivery system is composed of three functional domains assembled into a single recombinant molecule. The p21-derived peptide is positioned at the N-terminus and serves as the therapeutic cargo, functioning as a cyclin-dependent kinase (CDK) inhibitor that disrupts cell-cycle progression [8,9]. This domain is fused to thermally responsive ELP1, which acts as the biopolymer carrier and confers increased structural stability, improved pharmacokinetic persistence, and enhanced intracellular accumulation [6,7]. CPP (Bac) is appended to the C-terminus to promote cellular internalization and facilitate localization in proximity to the nuclear region [10].

By integrating these functional components into a single macromolecule, p21-ELP1-Bac enables efficient translocation across the plasma membrane and intracellular delivery of the p21-derived peptide to its site of action in glioblastoma cells, as seen in Figure 1.

2.2. Comparison of Antiproliferative Effects of p21-ELP1-Bac

The antiproliferative effects of p21-ELP1-Bac were evaluated in three glioblastoma cell lines (GBM43, U87, and GBM6) using a luminescent cell viability assay following 72 h of treatment (Figure 2). Cells were treated with increasing concentrations of p21-ELP1-Bac (5, 10, and 20 µM p21 equivalents), and cell viability was normalized to untreated controls. Treatment with p21-ELP1-Bac resulted in dose-dependent reductions in cell viability across all three glioblastoma models (

Table 1. Percent cell viability (mean ± SD) after 72 h treatment.

Dose/Treatment	GBM43	U87	GBM6
5 µM p21-ELP1-Bac	64 ± 4.6	73 ± 5.2	81 ± 5.4
10 µM p21-ELP1-Bac	55 ± 4.7	55 ± 4.8	74 ± 5.2
20 µM p21-ELP1-Bac	45 ± 5.3	40 ± 5.1	64 ± 4.9
IC50 values (µM) *	14.72 ± 3.3	12.90 ± 3.9	33.74 ± 6.8

Percent cell viability of GBM43, U87, and GBM6 cells following treatment with increasing concentrations of p21-ELP1-Bac (5, 10, and 20 µM). Viability was measured using the APExBIO luminescent cell viability assay and normalized to untreated controls. Data represent mean ± SD from n = 3 independent experiments, each performed with 3–6 technical replicates. Standard deviations (SD) had originally been rounded for simplicity. Unrounded standard deviation values ranged from approximately 4.6–5.4% across conditions. IC₅₀ values were calculated using a log(inhibitor) vs. response model. * Indicates IC₅₀ values (µM) derived from nonlinear regression analysis.

). At 20 µM, U87 cells showed the greatest sensitivity, with viability reduced to approximately 40%, followed by GBM43 cells (45%), while GBM6 cells were comparatively more resistant (64%). These findings indicate differential cytostatic responses to p21-ELP1-Bac across glioblastoma subtypes, with U87 cells exhibiting the strongest response under the conditions tested.

IC₅₀ values calculated from dose–response curves indicated that GBM6 (IC₅₀ 33.74 µM) cells required higher concentrations of p21-ELP1-Bac to achieve comparable growth inhibition relative to GBM43 (IC₅₀ 14.72 µM) and U87 (IC₅₀ 12.90 µM) cells.

2.3. Cellular Uptake of ELP-Delivered p21

Cellular uptake of p21-ELP1-Bac was assessed by flow cytometry using rhodamine-labeled conjugates following incubation for 1, 2, and 4 h at 10 µM p21 equivalents (Figure 3). All three glioblastoma cell lines demonstrated time-dependent increases in intracellular fluorescence relative to untreated controls.

These results underscore progressive intracellular accumulation of p21-ELP1-Bac in all three glioblastoma models, with lineage-specific differences in uptake kinetics and magnitude.

Flow cytometry-generated fluorescence intensity distributions of rhodamine-labeled p21-ELP1-Bac for each of the treatments were observed. GBM43 and U87 cells demonstrated rapid uptake, with mean fluorescence intensities increasing steadily from early to late time points. By contrast, GBM6 cells exhibited lower signal intensity and a delayed increase. Overall, intracellular fluorescence increased with incubation time in all three GBM cell lines.

2.4. Cell Cycle Distribution Following p21-ELP1-Bac Treatment

Inhibition of cyclin-dependent kinase activity is a key mechanism of p21 function, uniformly resulting in cell cycle arrest and decreased proliferation rate [11]. To compare the effects of p21 at specific stages of cell proliferation, cells were treated with p21 equivalents and analyzed by flow cytometry (Figure 4). Overall, p21-ELP1-Bac treatment did not induce statistically significant cell cycle arrest in any single phase across the three glioblastoma cell lines under the tested conditions, although intrinsic differences among GBM models were of note.

After 72 h of treatment with p21-ELP1-Bac, GBM43 cells exhibited a modest decrease in the G1 population with a corresponding increase in S-phase cells, while the G2 population remained largely unchanged compared to untreated controls. U87 cells showed a similar, slight redistribution from G1 into S phase; however, no statistically significant differences were observed across G1, S, or G2 phases. GBM6 cells displayed a comparatively high S-phase population under both treated and untreated conditions, and p21-ELP1-Bac treatment did not significantly alter cell cycle distribution in this cell line.

2.5. Intracellular Localization of p21-ELP1-Bac

The subcellular distribution of p21-ELP1-Bac was determined by confocal microscopy (Figure 5). Confocal imaging was performed over a short incubation period (≤1 h), during which no obvious acute cytotoxic effects, such as cell rounding or detachment, were observed at 20 µM. Overlay images revealed fluorescence near the nuclear region in GBM43, U87, and GBM6 cells. The punctate intracellular fluorescence observed likely reflects intracellular compartmentalization and local accumulation of the ELP product; quantitative assessment of macromolecular stability or aggregation was beyond the scope of this study. These observations were qualitative in nature and were reproducible across replicate experiments.

2.6. Apoptotic Response to p21-ELP1-Bac

Induction of apoptosis by p21-ELP1-Bac was evaluated by flow cytometry following 24 h p21-ELP1-Bac treatment (Figure 6). GBM43, U87, and GBM6 cells were analyzed using a double-staining Annexin V/PI assay to assess apoptotic responses to treatment. Etoposide-treated cells served as positive controls and repeatedly demonstrated apoptotic induction. Under these conditions, GBM43 and U87 cells showed minimal apoptotic response, whereas GBM6 cells exhibited increases in early and late apoptotic populations as well as necrosis. The percentage of early apoptotic cells following treatment with p21-ELP1-Bac was approximately 2% in GBM43 cells, 8% in U87 cells, and 5% in GBM6 cells.

3. Discussion

In this study, we investigated the biological effects of delivery-enabled intracellular administration of a p21-derived peptide using an elastin-like polypeptide (ELP) carrier across three glioblastoma (GBM) cell lines. Treatment with p21-ELP1-Bac resulted in dose-dependent suppression of cell proliferation in GBM43, U87, and GBM6 cells, while revealing clear lineage-specific differences in cell-cycle regulation and apoptotic susceptibility. Importantly, the primary objective of this work was to evaluate intracellular p21 activity following delivery, as previous studies have highlighted the limited efficacy of unmodified p21 peptides in glioblastoma and other cancer models due to poor cellular uptake [12].

Although efficient cellular uptake of the protein-based polymer was observed in all three models, distinct biological responses emerged. GBM43 and U87 cells primarily exhibited growth suppression with minimal induction of apoptosis, substantiating a predominantly cytostatic response. In contrast, GBM6 cells demonstrated relative resistance to proliferation inhibition but showed increased susceptibility to apoptosis. These findings highlight distinct biological responses among glioblastoma subtypes and provide context for the mechanistic discussion below.

ELPs have emerged as versatile macromolecular carriers for intracellular drug delivery due to their favorable physicochemical properties and compatibility with engineered drug delivery systems. The p21-ELP1-Bac system integrates a p21-derived inhibitory peptide, a thermally responsive ELP carrier, and a C-terminal cell-penetrating peptide (Bac), enabling efficient cellular internalization and nucleus-associated localization. Under the experimental conditions used in this study, the delivery-enabled therapeutic exhibited minimal aggregation, and rhodamine conjugation remained stable throughout imaging experiments, ensuring reliable visualization of intracellular trafficking and treatment localization.

Building on prior work demonstrating the efficacy of ELP-mediated delivery of p21-derived peptides in other cancer models, this study extends the approach to glioblastoma. Evaluation across multiple GBM cell lines enabled direct comparison of lineage-specific responses and provided insight into how intrinsic cellular differences influence sensitivity to p21-mediated growth suppression [7,13]. Notably, GBM stem-like populations have been reported to exhibit impaired G1–S checkpoint regulation and sustained proliferative signaling, which may explain our observation of minimal G1 accumulation and dominant S-phase population retention in GBM6 cells after p21 treatment [14]. Despite this relative resistance, measurable growth inhibition at higher concentrations indicates that p21-ELP1-Bac retains antiproliferative activity even in treatment-resistant subpopulations.

Concordant with established cytostatic functions of p21, modest but reproducible shifts in cell-cycle distribution were observed [8,9]. In U87 and GBM43 cells, treatment induced moderate accumulation in the G1 phase, accompanied by a reduction in S-phase cells, indicative of CDK2 inhibition. In contrast, GBM6 cells displayed subtler changes, reflecting a high baseline S-phase fraction and suggesting altered replication dynamics or compensatory signaling pathways that attenuate the impact of exogenous p21.

Cellular uptake analyses further supported p21-ELP1-CPP functionality, demonstrating time-dependent intracellular accumulation across all cell lines. A concentration of 20 µM was selected based on prior uptake studies to ensure sufficient fluorescence detectability, and imaging revealed no evidence of acute cytotoxic morphology or loss of adherence. These observations indicate that the observed growth suppression and downstream cellular responses are not attributable to nonspecific toxicity. Confocal imaging was noted to demonstrate fluorescence localized near the nucleus across all concentrations and models, along with partial cytoplasmic localization. The observed intracellular fluorescence pattern reflects the distribution of the ELP construct within cytoplasmic and nuclear-associated compartments rather than exclusive nuclear localization. This localization pattern aligns with prior reports showing sustained intracellular retention and functional nuclear engagement of CPP–ELP conjugates [10].

Short p21-derived peptides lack intrinsic cell-penetrating motifs and are not expected to cross cellular membranes efficiently. In prior studies using ELP1 delivery platforms, intracellular uptake was shown to be dependent on the presence of CPPs. Whereas molecular platforms lacking CPPs exhibited minimal cellular internalization [6,10,15].

Previous work from our group demonstrated that incorporation of cell-penetrating peptides, including Bac and related CPPs, significantly enhances delivery of ELP-based polypeptides to intracranial glioma tumors, resulting in increased tumor accumulation and therapeutic efficacy compared with non-CPP controls [10,15]. This platform has been used to deliver both peptide-based therapeutics, such as a c-Myc inhibitory peptide, as well as small-molecule agents, including doxorubicin, in glioblastoma-relevant in vivo models [15,16]. Based on these established behaviors, the objective of the present work was to characterize the ELP-CPP-mediated intracellular activity and cytostatic effects of delivery-enabled p21 following cellular uptake, rather than to re-evaluate blood–brain barrier penetration or carrier performance, which have been investigated in prior studies using related ELP-CPP delivery systems [15].

The lineage-specific responses observed across GBM cell lines further emphasize the biological heterogeneity of glioblastoma and highlight the importance of considering tumor-intrinsic differences when evaluating peptide-based delivery strategies. To our knowledge, no in vitro blood–brain barrier penetration studies of p21 or p21-derived biopolymers have been reported; thus, establishing intracellular activity in glioblastoma cells outside a BBB context represents a necessary first step. While the present work provides proof-of-concept for intracellular p21 activity, future investigations should address BBB penetration, in vivo efficacy, and therapeutic durability. Extension of this approach to glioblastoma animal models, leveraging the thermally responsive properties of ELP carriers for localized tumor accumulation, and evaluating combinatorial treatment strategies with chemotherapeutics, may further enhance therapeutic potential. Additionally, transcriptomic and proteomic analyses could help identify molecular determinants of sensitivity or resistance to p21-mediated growth suppression, supporting more targeted treatment strategies.

In summary, these findings provide strong evidence that ELP-based delivery of p21 represents a viable strategy for suppressing glioblastoma cell proliferation. The p21-ELP1-Bac system demonstrates efficient cellular uptake, stable fluorescent labeling, near nuclear localization, and CDK-mediated growth suppression that is largely independent of apoptosis. Together with the modular and adaptable nature of the ELP delivery platform, these results support further exploration of p21-ELP1-Bac as a component of multimodal therapeutic strategies for aggressive brain tumors.

4. Materials and Methods

4.1. Polypeptide Expression and Purification

Construct design, expression, and purification of ELP-based polypeptides were performed as previously described by our group, including iterative, directional cloning for ELP synthesis, bacterial expression in E. coli, and purification by inverse thermal cycling. The p21-ELP1-Bac construct used in this study employs an elastin-like polypeptide carrier similar to those characterized by Liu et al. (2006) [12] and incorporates a p21-derived inhibitory peptide and Bac cell-penetrating sequence as described in our prior laboratory studies. The full amino acid sequence of the engineered polypeptide is:

GRKRRQTSMTDFYHSKRRLIFSKRKP-GCGPGVG-(VPGXG)₁₅₀-RRIRPRPPRLPRPRPRPLPFPRP.

The N-terminal p21-derived peptide (GRKRRQTSMTDFYHSKRRLIFSKRKP, 26 amino acids) corresponds to a C-terminal region of p21 previously shown to inhibit cyclin-dependent kinase activity [5,6,8]. This peptide was linked to the elastin-like polypeptide carrier via a short GCGPGVG linker. The ELP domain consisted of 150 repeats of the pentapeptide motif (VPGXG), where X represents a mixture of valine, glycine, and alanine in a 5:3:2 ratio. A C-terminal Bac cell-penetrating peptide (RRIRPRPPRLPRPRPRPLPFPRP, 23 amino acids) was included to facilitate cellular uptake and intracellular delivery.

4.2. Cell Lines

GBM43, U87, and GBM6 glial cancer cells were obtained from Translational Neuro-Oncology: Dr. Jann N. Sarkaria—Mayo Clinic Research Lab, Scottsdale, AZ, USA and were handled according to institutional guidelines. Cells were maintained at 37 °C with 5% CO₂ and 95% humidity in DMEM (Corning Inc., Corning, NY, USA) supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, Georgia, USA) and 1% penicillin–streptomycin (HyClone Laboratories, Logan, UT, USA). Cells were sub-cultured at 80% confluence using 0.25% Trypsin every 3–5 days, to maintain logarithmic growth. No human participants or animal subjects were directly involved in this study.

4.3. Antiproliferative Assay

APExBIO luminescent assay (Promega, Fitchburg (Madison), WI, USA) was used to assess cell viability following antiproliferative treatment with p21-ELP1-Bac. Cells (1 × 10³ per well) were seeded in opaque 96-well plates in triplicate, allowed to adhere overnight, and treated for 72 h with two-fold increasing concentrations of p21-ELP1-Bac. Untreated cells served as controls. Luminescence was measured on a Synergy H4 plate reader (Agilent Technologies, Santa Clara, CA, USA), and cell viability was expressed as a percentage of untreated controls. Standard deviations (SD) had originally been rounded for simplicity. Unrounded standard deviation values ranged from approximately 4.6–5.4% across conditions.

4.4. Apoptosis Assay

Annexin V-Alexa 488 and Propidium Iodide (PI) (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were used to assess drug-induced apoptosis, then measured by flow cytometry using a NovoCyte Flow Cytometer (Agilent, Santa Clara, CA, USA). GBM43, U87, and GBM6 cells (2.25 × 10⁵/well) were seeded in 6-well plates, incubated overnight, and treated for 24 h with p21-ELP1-Bac. Etoposide (500 µM) was used as a positive control for apoptosis. After treatment, floating and adherent cells were collected, stained with Annexin V Alexa 488 and propidium iodide (PI), and analyzed. Early apoptosis, late apoptosis, and necrosis were quantified based on Annexin V and PI fluorescence.

4.5. Cell Cycle Analysis

GBM43, U87, and GBM6 cells (5 × 10⁵/well) were seeded in 6-well plates and treated for 72 h with p21-ELP1-Bac. After treatment, cells were washed with PBS, fixed in 70% ethanol (on ice) for 30 min, rinsed, and resuspended in PBS. To eliminate RNA interference, 30 µL of 10 mg/mL RNase A was added to a final concentration of 750 µg/mL. Cells were stained with PI (150 µL) for 30 min at room temperature, and DNA content was analyzed using a NovoCyte flow cytometer. GraphPad software 10.6.1 was used to assess cell cycle distribution. Python software 3.12 was used to enhance visibility of qualitative data.

4.6. Cellular Uptake Assay

GBM43, U87, and GBM6 cells (8 × 10⁵/well) were seeded in 6-well plates, incubated overnight, and treated at 1-, 2-, and 4-h increments with p21-ELP1-Bac. Cells were harvested using 0.05% Trypsin (Thermofisher Scientific, Inc., Waltham, MA, USA), and intrinsic fluorescence was quantified by flow cytometry (20,000 events/sample). Gating was selective to exclude aggregate data and normalize to autofluorescence.

4.7. Cellular Localization

To visualize the intracellular localization of p21-ELP1-Bac delivery in all three cell lines, cells were seeded at 70% confluence on 35 mm Petri dishes. The 20 µM dose was selected to maximize intracellular detectability without inducing acute cytotoxicity or loss of adherence. After 24 h, cells were treated for 1 h with Invitrogen tetramethylrhodamine-5-maleimide labeled p21-ELP1-Bac, washed with PBS, fixed in cold methanol, and stained with CyQuant/Sytox Green (Thermo Fisher Scientific). Rhodamine was covalently conjugated to the ELP-based system, and unbound dye was removed during purification. Petri dishes were mounted and imaged using a Nikon confocal microscope (Nikon Instruments Inc., Shinagawa, Tokyo). This data is qualitative only.

4.8. Statistical Analysis

GraphPad Prism 10.6.1 was used to plot and statistically analyze viability data. Standard deviation (SD) was calculated from at least three independent experiments. One-way ANOVA was used to assess statistical significance among treatment groups and controls.

5. Conclusions

Our findings show that elastin-like polypeptide–mediated delivery of a p21-derived peptide reliably produces antiproliferative effects across multiple glioblastoma cell models. The p21-ELP1-Bac delivery system was efficiently internalized by all three GBM cell lines and demonstrated nuclear-targeted localization, accompanied by growth suppression without widespread induction of apoptosis. While clear lineage-specific differences were observed—most notably the relative resistance and altered cell cycle profile of GBM6—p21-ELP1-Bac reduced proliferation in all models examined, including those with intrinsic cell cycle dysregulation. Together, these data position p21-ELP1-Bac as a promising cytostatic platform for GBM therapy and support its further evaluation in vivo, where ELP thermal targeting and conjunctive approaches may amplify therapeutic benefit.