Comparative Study of Polymer-Based Thermo-Responsive Systems for Enhanced Doxorubicin Delivery in Tumor-Specific Chemotherapy

Cancer remains one of the leading causes of morbidity and mortality worldwide, characterized by uncontrolled cellular proliferation and the potential to invade distant organs. Despite considerable advances in understanding tumor biology, chemotherapy continues to be a cornerstone in cancer treatment [1]. However, conventional chemotherapy is fraught with challenges such as systemic toxicity, development of multidrug resistance, and lack of tumor specificity. These drawbacks significantly limit therapeutic efficacy and impair patients' quality of life. Chemotherapeutic drugs cannot often distinguish between malignant and healthy cells, leading to widespread damage to rapidly dividing normal tissues such as the gastrointestinal tract, bone marrow, and hair follicles. Moreover, the need for repeated administration of high drug doses contributes to cumulative toxicity, organ dysfunction, and sometimes irreversible damage. As cancer therapy moves toward personalized and targeted approaches, the inadequacies of conventional chemotherapy necessitate the development of more intelligent and efficient drug delivery strategies [2]. Doxorubicin (DOX), an anthracycline antibiotic, is one of the most widely used chemotherapeutic agents in treating a variety of cancers including breast cancer, leukemia, and lymphomas. Its mechanism of action involves intercalation into DNA, inhibition of topoisomerase II, and the generation of reactive oxygen species (ROS), collectively leading to apoptosis of cancer cells [3]. While DOX is highly effective, its clinical use is limited by dose-dependent toxicities, particularly cardiotoxicity, which can progress to congestive heart failure. The heart, due to its limited antioxidant capacity, becomes especially vulnerable to ROS generated during DOX metabolism. In addition to cardiotoxicity, DOX is associated with other adverse effects such as myelosuppression, mucositis, alopecia, and hepatotoxicity. Furthermore, the emergence of multidrug resistance (MDR), primarily through the overexpression of efflux transporters like P-glycoprotein, significantly reduces the intracellular concentration of DOX, undermining its therapeutic efficacy. These challenges underscore the urgent need for alternative delivery methods that enhance drug accumulation in tumors while minimizing systemic exposure [4]. To overcome the limitations of traditional chemotherapy, research has increasingly focused on the design of smart drug delivery systems (SDDSs) that respond to specific biological or external stimuli to release the therapeutic agent in a controlled and targeted manner. Such systems aim to maximize drug concentration at the tumor site while sparing normal tissues, thereby improving the therapeutic index of anticancer agents [5]. Smart drug delivery platforms are often engineered to respond to various stimuli including pH, enzymes, redox conditions, and temperature conditions that are typically altered in the tumor microenvironment. Among these, thermo-responsive systems have gained significant attention due to their relatively straightforward design and external controllability. These systems can be activated by localized hyperthermia (e.g., through infrared or magnetic heating), which is often used in cancer treatment as an adjuvant therapy to improve perfusion and oxygenation of tumor tissues [6]. Thermo-responsive polymers are a class of "intelligent" materials that undergo a reversible phase transition in response to changes in temperature. These polymers exhibit a critical solution temperature, typically the lower critical solution temperature (LCST), below which they are soluble in aqueous media and above which they undergo phase separation. This thermal sensitivity enables them to act as "on-off" switches for drug release [7]. One of the most commonly studied thermo-responsive polymers is poly(N-isopropylacrylamide) (PNIPAM), which has an LCST close to physiological temperature (~32 °C). Above this temperature, PNIPAM becomes hydrophobic and collapses, leading to the expulsion of water and entrapped drugs. This behavior can be harnessed to trigger the release of chemotherapeutic agents specifically at the tumor site, where the local temperature is elevated due to hyperthermia or inflammatory responses [8].

The key advantages of thermo-responsive systems include:

• Controlled and sustained drug release at the desired site and time,
• Reduced systemic toxicity through site-specific delivery,
• Minimal invasiveness, particularly when triggered by external stimuli,
• Improved drug stability and bioavailability.

Such systems can be tailored by modifying polymer composition, architecture, and crosslinking density to achieve desired release kinetics and compatibility with the encapsulated drug [9]. Given the promising potential of thermo-responsive polymers for targeted drug delivery, this study aims to perform a comparative evaluation of different polymer-based thermosensitive systems for the enhanced delivery of doxorubicin in tumor-specific chemotherapy. The polymers investigated include PNIPAM-based hydrogels, PLGA-PNIPAM-PEG copolymeric nanoparticles, and other hybrid systems incorporating thermoresponsive and biodegradable components Each system was evaluated for its physicochemical characteristics, drug loading capacity, in vitro drug release behavior, biocompatibility, and cytotoxic efficacy against cancer cell lines. Special emphasis was placed on assessing the temperature-triggered release profiles and retention of antitumor activity under simulated physiological and hyperthermic conditions. In addition, the study explored the potential of these systems to mitigate the cardiotoxic effects of DOX by reducing its systemic exposure and enhancing tumor specificity. By systematically comparing multiple polymeric carriers, this study contributes to the growing field of smart chemotherapy delivery and provides insights into the design parameters that govern the efficiency, responsiveness, and safety of thermo-responsive nanocarriers. Ultimately, the findings aim to support the development of next-generation drug delivery platforms that can significantly improve cancer treatment outcomes. In cancer therapy, tumor tissues often exhibit a higher local temperature (about 40–42°C) compared to normal tissues, owing to their abnormal vasculature and metabolic activity. Thermo-responsive polymers leverage this gradient to achieve spatial and temporal control of drug release. The key feature of these systems is the lower critical solution temperature (LCST)—a temperature above which the polymer undergoes a sol-gel transition or collapse that facilitates the release of encapsulated drugs. Polymers designed with LCSTs around 37–42°C are ideal for tumor-specific applications (Lacroce, 2022) [11].

1. 1. Thermo-Responsive Polymers in Cancer Therapy

Several thermo-responsive polymers have been explored for drug delivery, each with unique physicochemical properties, responsiveness, and drug compatibility. Notable among these are poly(N-isopropylacrylamide) (PNIPAM), Pluronic block copolymers, and poly(ethylene glycol)-based (PEG-based) systems (Jia et al., 2021) [12].

1. 2. PNIPAM-Based Systems

PNIPAM is one of the most studied thermo-responsive polymers, characterized by an LCST around 32°C, which can be tuned by copolymerization or functionalization (Metawea et al., 2021) [13]. PNIPAM undergoes a reversible coil-to-globule transition near its LCST, allowing efficient encapsulation and release of hydrophilic and hydrophobic drugs. Studies by Xin et al. (2022) demonstrated that PNIPAM hydrogels loaded with doxorubicin released the drug rapidly when the temperature was raised above the LCST, resulting in enhanced cytotoxicity against breast cancer cell lines [14]. Modifications such as grafting PNIPAM with polyacrylic acid or PEG can enhance its biocompatibility and tailor its responsiveness. However, concerns over PNIPAM’s in vivo degradation and potential accumulation have led researchers to explore copolymer blends and hybrid systems to improve safety and clearance (Khan, 2019) [15].

1. 3. Pluronic-Based Systems

Pluronics (also known as poloxamers) are triblock copolymers composed of poly (ethylene oxide)–poly (propylene oxide)–poly (ethylene oxide) (PEO–PPO–PEO). These amphiphilic molecules form micelles at physiological temperatures and exhibit a Sol-gel transition at elevated temperatures, making them excellent thermo-sensitive carriers. Pluronic F127, in particular, has been studied extensively for doxorubicin delivery. For example, Cheng et al. (2020) reported that Pluronic micelles enhanced drug accumulation in multidrug-resistant tumors by disrupting cellular efflux mechanisms and providing sustained release [16]. Pluronics are advantageous due to their FDA-approved status, ease of formulation, and synergistic effect with chemotherapeutic agents. However, their relatively low mechanical strength and burst release tendencies necessitate blending with other polymers or crosslinkers.

1. 4. PEG-Based and Hybrid Systems

PEG-based polymers are often used to enhance hydrophilicity and circulation time of drug carriers. While PEG itself is not thermo-responsive, its copolymerization with thermo-sensitive monomers such as PNIPAM or polycaprolactone (PCL) results in stimuli-sensitive copolymers with improved stability (Wang et al., 2023) [17] Hybrid systems such as PEG–PNIPAM or PEG–PCL have shown promising results in in vitro and in vivo studies. For instance, Mohajeri et al., (2024) demonstrated that PEG–PNIPAM nanoparticles offered enhanced drug loading, prolonged circulation, and tumor-specific accumulation of doxorubicin [18]. PEGylation also reduces opsonization and clearance by the mononuclear phagocyte system (MPS), enabling passive tumor targeting via the enhanced permeability and retention (EPR) effect.

1. 5. Doxorubicin Encapsulation and Release Profiles

Doxorubicin (DOX), a potent anthracycline antibiotic used in various cancers, suffers from limitations including cardiotoxicity, non-specific distribution, and development of drug resistance. Encapsulating DOX in thermo-responsive systems can mitigate these drawbacks by allowing for triggered release at tumor sites, reducing systemic toxicity (Jones, 2022) [19].

1. 6. Gaps in Current Research and the Need for Comparative Evaluation

Lack of standardized comparative studies: Most studies focus on a single polymer system in isolation. Few directly compare multiple thermo-responsive systems under identical experimental conditions for parameters such as encapsulation efficiency, release kinetics, cytotoxicity, and thermal responsiveness.

Insufficient understanding of structure-function relationships: The influence of polymer architecture, crosslinking density, and surface modification on drug release and biocompatibility is not fully understood.

Limited translational insight: While many formulations demonstrate promise in vitro, in vivo behavior including biodistribution, toxicity, and clearance remain poorly explored.

Variable methodologies: Diverse preparation techniques and assay conditions make cross-study comparisons difficult. The present study aims to address these limitations by conducting a systematic, side-by-side comparison of three representative thermo-responsive polymer systems—PNIPAM, Pluronic F127, and PEG-based copolymers—for the encapsulation and controlled release of doxorubicin. Through comprehensive in vitro evaluation—including physicochemical characterization, temperature-responsive behavior, and cytotoxicity assays—this study seeks to identify the most promising system for future in vivo and clinical applications in tumor-specific chemotherapy.

2. MATERIALS AND METHODS

2.1 Study Design

This experimental study was designed to develop and evaluate polymer-based thermoresponsive drug delivery systems for targeted doxorubicin delivery in tumor-specific chemotherapy. The research comprised three major phases: (1) formulation and optimization of thermoresponsive carriers, (2) characterization and in vitro drug release studies, and (3) assessment of cytotoxicity and biocompatibility.

2.2 Materials and Equipment

All reagents were of analytical or pharmaceutical grade. Doxorubicin hydrochloride was procured from Cipla Ltd. or equivalent certified sources. Polymers and other chemicals were obtained from Sigma-Aldrich, Merck, HiMedia, and SRL Pvt. Ltd. Instruments used were calibrated prior to experimentation. Tables 1 and 2 summarize the materials and equipment.

2.3 Formulation and Preparation

2.3.1 Synthesis of Thermoresponsive Polymers

• PNIPAM Hydrogels were synthesized via free-radical polymerization using NIPAM, BIS (cross-linker), and APS (initiator) under nitrogen at 25°C for 2 hours. Unreacted monomers were removed by dialysis, and the resulting hydrogels were lyophilized for further use.
• Pluronic F127 Micelles were formed via the cold method: F127 was dissolved in PBS (4°C, overnight), followed by spontaneous micelle formation at 37°C and purification via centrifugation.

2.3.2 Encapsulation of Doxorubicin

PNIPAM Hydrogels: DOX was dissolved in PBS, mixed with the hydrogel at 4°C, and incubated at 37°C for gelation and entrapment. Excess drug was removed by washing and lyophilization.

Pluronic F127 Micelles: DOX was added dropwise to the micellar solution, stirred at 4°C for 3 hours, and stabilized at 37°C. The free drug was removed via dialysis (MWCO 3.5 kDa, 12 hours).

Encapsulation Efficiency (EE) and Drug Loading (DL) were calculated as:

2.3.3 Characterization of Formulations

Characterization included particle size, polydispersity index, zeta potential (via DLS), morphology (via SEM), and structural integrity (via FTIR and NMR). Thermal behaviour and gelation properties were examined using DSC and visual observation.

2.4 In Vitro Drug Release Studies

Drug release was assessed under physiological (37°C) and hyperthermic (42°C) conditions using dialysis bag diffusion method in PBS (pH 7.4 and 6.5). Cumulative release was quantified using HPLC and analyzed via multiple kinetic models (Zero-order, First-order, Higuchi, Korsmeyer–Peppas) to determine release mechanisms.

2.5 Stability Studies

Formulations were stored under both accelerated and long-term conditions. At defined intervals, samples were analyzed for physical appearance, particle size, drug content, and thermal stability. Stability was inferred based on ICH guidelines.

2.6 Cytotoxicity and Biocompatibility

2.6.1 MTT Assay

MCF-7, A549, and fibroblast cell lines were used to assess cytotoxicity via MTT assay. Cells were exposed to free DOX, DOX-loaded carriers, and blank polymers. IC?? values were calculated and compared to evaluate enhanced cytotoxicity against cancer cells and minimal toxicity to normal cells.

2.6.2 Hemocompatibility Testing

Hemocompatibility of the formulations was assessed using fresh human blood obtained with informed consent, following institutional ethical guidelines. Red blood cells (RBCs) were isolated by centrifugation and incubated with test formulations for 1 hour at 37°C. Following incubation, samples were centrifuged, and the supernatant was analysed for haemoglobin release using a UV-Vis spectrophotometer at 540 nm.

Hemolysis (%) was calculated using the following equation:

• Negative control: PBS (baseline hemolysis)
• Positive control: 1% Triton X-100 (100% hemolysis)

A hemolysis value of less than 5% was considered indicative of acceptable hemocompatibility according to ASTM F756-17 standards.

2.7 Statistical Analysis

All experiments were conducted in triplicate. Data were expressed as mean ± standard deviation (SD). Statistical comparisons were performed using one-way ANOVA followed by Tukey’s post-hoc test. A p-value < 0.05 was considered statistically significant. GraphPad Prism 9.0 software was used for data analysis and graph generation.

3. RESULT

The results of the study on the development and evaluation of polymer-based thermoresponsive systems for enhanced doxorubicin delivery are presented below, with a focus on key aspects such as encapsulation efficiency, drug loading, characterization of the polymeric formulations, temperature-triggered release profiles, and comparative cytotoxicity data.

3.1 Encapsulation Efficiency and Drug Loading

The encapsulation efficiency (%EE) and drug loading (%DL) of doxorubicin in various polymeric formulations were evaluated across different polymer concentrations. The encapsulation efficiency is crucial in assessing the drug retention capability of the polymeric system, while the drug loading reflects the weight percentage of the drug within the final formulation.

The results revealed that increasing polymer concentration significantly improved both encapsulation efficiency and drug loading. Formulation F4, which contained the highest polymer concentration, exhibited the highest encapsulation efficiency (88.1%) and drug loading (24.1%). Statistical analysis confirmed a highly significant effect of polymer concentration on encapsulation efficiency (F = 232.06, p = 0.000005). This indicates that higher polymer concentrations provide a more stable matrix for doxorubicin entrapment, reducing drug leakage and ensuring better drug retention.

Fig 1: Encapsulation Efficiency & Drug Loading

3.2 Characterization Results

The physicochemical properties of the synthesized polymeric formulations were thoroughly analyzed to confirm successful synthesis, encapsulation, and drug-polymer interactions.

3.2.1 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR analysis was performed to assess the functional groups and confirm drug-polymer interactions. The FTIR spectrum of PNIPAM, the blank polymer, showed characteristic absorption peaks at 1650 cm?¹ (C=O stretching) and 3300 cm?¹ (N-H stretching), indicative of the polymer's amide groups. After drug loading, slight shifts in these peaks (1645 cm?¹ and 3285 cm?¹) were observed, suggesting hydrogen bonding between the carbonyl group of PNIPAM and the hydroxyl groups of doxorubicin. These spectral changes support the hypothesis that drug-polymer interactions contribute to the stability of the doxorubicin-loaded formulations.

3.2.2 Differential Scanning Calorimetry (DSC)

DSC thermograms were used to assess the thermal properties and confirm drug-polymer interactions. The blank polymer showed a glass transition temperature (Tg) of 60°C, typical for PNIPAM. In contrast, the doxorubicin-loaded formulation displayed a Tg of 72°C, indicating restricted polymer chain mobility due to drug incorporation. Additionally, an exothermic peak at 90°C, unique to the drug-loaded formulation, suggested the crystalline nature of doxorubicin within the polymer matrix, further supporting the successful encapsulation.

3.2.3 Nuclear Magnetic Resonance (NMR) Spectroscopy

Proton NMR spectra confirmed the structural integrity of the polymer and the successful encapsulation of doxorubicin. Peaks at δ1.1–1.4 ppm corresponding to PNIPAM's polymer backbone and peaks at δ7.2–7.8 ppm corresponding to doxorubicin's aromatic protons were observed in the drug-loaded formulation, indicating that both the polymer and drug were present in the final formulation without significant chemical modifications.

3.3 Temperature-Triggered Release Profiles

The temperature-triggered release behavior of doxorubicin from the polymeric formulations was studied under physiological conditions (pH 7.4) and tumor-mimicking conditions (pH 6.5, 42°C). The results were compared to free doxorubicin, which exhibited a rapid burst release.

At 1 hour, free doxorubicin released 30.1%, whereas polymeric formulations released between 6.2% (F4) and 10.5% (F1), indicating a slower release. At 24 hours, free doxorubicin exhibited near complete release (98.1%), while polymeric formulations showed a sustained release ranging from 60.5% to 78.9%. These results suggest that the polymeric formulations offer controlled drug release, which is crucial for achieving sustained therapeutic effects.

Graph 2: In Vitro Drug Release Profiles of Doxorubicin from Polymeric Formulations and Free Doxorubicin

3.4 Comparative Cytotoxicity Data

Cytotoxicity was evaluated using an MTT assay on cancer cell lines to assess the effectiveness of the polymeric formulations in inhibiting cell growth. The results demonstrated that polymeric formulations provided controlled cytotoxicity, avoiding the rapid and aggressive toxicity associated with free doxorubicin.

At lower concentrations (0.1 µg/mL), the cell viability was above 85% for all polymeric formulations, whereas free doxorubicin reduced cell viability to 76.2%. As the concentration increased, the polymeric formulations showed more controlled cytotoxicity, with cell viability ranging from 22.8% to 92.3%, while free doxorubicin drastically reduced viability (5.8% at 5.0 µg/mL). This suggests that the polymeric formulations allow for gradual drug release, minimizing sudden high toxicity while maintaining effectiveness.

Graph 3: MTT Assay – Comparative Cytotoxicity Data for Various Formulations