The central insight behind the work is that resistance to doxorubicin is not a single mechanism but a coordinated defense. P-glycoprotein, an ATP-dependent efflux transporter embedded in the membranes of many cancer cells, actively pumps the drug back out, keeping intracellular concentrations below lethal thresholds. Meanwhile, CD44, a cell surface glycoprotein implicated in adhesion, invasion, metastasis and the survival of cancer stem cells, helps maintain a reservoir of therapy-refractory cells that can seed recurrence after treatment ends. Reserpine was selected as a chemosensitizer because it can potentially silence both pathways simultaneously. Before any formulation work began, the team used AutoDock Vina molecular docking to test this hypothesis computationally, comparing reserpine against verapamil, a clinically established P-glycoprotein inhibitor used as a reference ligand.

The docking results were striking. Reserpine bound P-glycoprotein with a predicted affinity of −9.07 kcal/mol, substantially stronger than verapamil’s −7.10 kcal/mol, forming hydrogen bonds with residues including Lys287, Lys822 and Arg785 while its aromatic rings and methoxy groups anchored the molecule through hydrophobic and pi-stacking contacts with Phe766, Val831 and Ala288, among others. Against CD44, reserpine scored −7.46 kcal/mol versus verapamil’s −6.34 kcal/mol, stabilized by hydrogen bonds to Asn100, Ser95 and Ser109 and hydrophobic interactions with Ala99, Tyr79 and Ile96. These in silico findings, the authors note, provide the first mechanism-based computational evidence that reserpine can simultaneously target P-gp mediated drug efflux and CD44-mediated tumor stemness, positioning it as a rational partner for doxorubicin rather than merely an additive.

With the biological rationale established, the formulation challenge was to build a carrier that would keep the drug pair confined to the tumor. The team turned to poloxamers, triblock copolymers of poly(ethylene oxide) and poly(propylene oxide) that are FDA-accepted, biocompatible and famous for a reversible sol-gel transition near body temperature. Using a cold method, the researchers dissolved poloxamer 407 and poloxamer 188 in chilled phosphate buffer, allowed full hydration over 24 hours at 4 °C, and then loaded doxorubicin into one portion of the polymer solution while reserpine, aided by a small amount of Tween 80, was sequestered into the hydrophobic cores of poloxamer micelles in the other. The two streams were combined at a 5:1 doxorubicin-to-reserpine weight ratio, yielding a final concentration of 2 mg/mL doxorubicin and 0.4 mg/mL reserpine in a clear, homogeneous sol.

Three candidate formulations were screened, and the winner, designated F2, contained 22% poloxamer 407 and 15% poloxamer 188. Its physicochemical profile read like a checklist for intratumoral injection: a pH of 5.5 to 6.1 compatible with the acidic tumor microenvironment, low viscosity of 380 to 460 centipoise at refrigeration temperature for easy handling, higher but still injectable viscosity of 1320 to 1580 centipoise at 25 °C, syringeability times of 4 to 7 seconds through an 18-gauge needle, and a rapid sol-gel transition between 38.5 and 41.2 °C with gelation times of just 37 to 42 seconds. Gel strength, measured by a modified falling-weight test, ranged from 47 to 56 seconds, sufficient to maintain structural integrity at the injection site. Drug content was high at 94 to 96 percent, and entrapment efficiency peaked at 91.6 percent for F2, confirming that both drugs were retained efficiently within the matrix.

Spectroscopic and thermal analyses then probed what had happened to the drugs inside the gel. Fourier transform infrared spectroscopy preserved all characteristic peaks of doxorubicin, reserpine and both poloxamers in the finished formulation, with only minor shifts, indicating physical encapsulation without chemical interaction. Differential scanning calorimetry told a more dramatic story: the sharp melting endotherms of crystalline doxorubicin at 180.4 °C and crystalline reserpine at 274.5 °C vanished or were greatly reduced in the loaded gel, leaving only a broad poloxamer transition near 54.7 °C. X-ray diffraction corroborated this, showing that the distinctive sharp peaks of both drugs disappeared entirely upon loading, replaced by the broad halos of the semicrystalline polymers. Together, these data demonstrate that both drugs exist in an amorphous, molecularly dispersed state within the gel matrix, a form that generally favors improved solubility and release behavior.

Microscopy and light scattering filled in the nanoscale picture. Dynamic light scattering measured a mean hydrodynamic particle diameter of 90.22 nanometers with a polydispersity index of 0.492, placing the carriers firmly in the size range associated with good tumor tissue penetration. A zeta potential of −32.6 millivolts indicated strong electrostatic repulsion between particles, enough to prevent aggregation during storage. Scanning electron micrographs at 370-fold and 600-fold magnification revealed an irregular, porous, interconnected network of cavities and channels, exactly the architecture expected to admit water, swell gradually and release drug by diffusion through hydrated pathways rather than in a sudden burst. Three-month stability studies at both 4 to 8 °C and 25 °C with 65 percent relative humidity confirmed the formulation remained clear and homogeneous, with only slight viscosity drift and no loss of entrapment efficiency.

Release behavior proved to be the system’s most elegant feature. In dialysis experiments run at 38 °C, the gel released only 36 percent of its doxorubicin and 28 percent of its reserpine over 24 hours at physiological pH 7.4, but at pH 5.5, mimicking the acidic tumor microenvironment, release jumped to 96 percent for doxorubicin and 82 percent for reserpine. Because poloxamers lack intrinsic pH-responsive groups, the authors attribute this selectivity to temperature-driven micellization and polymer relaxation combined with pH-dependent changes in drug ionization and solubility. Kinetic modeling showed the Korsmeyer-Peppas equation fit best, with R² values of 0.91 to 0.96 and release exponents of 0.65 to 0.70, consistent with non-Fickian anomalous transport governed by both diffusion and polymer relaxation. The biological payoff came in MTT assays against MCF-7 breast cancer cells: the dual-drug gel achieved an IC₅₀ of 21.8 µg/mL, roughly half that of free doxorubicin at 42.6 µg/mL and a quarter that of free reserpine at 84.3 µg/mL, while the blank gel left more than 90 percent of cells viable, confirming carrier biocompatibility.

Safety data from an acute toxicity study in Swiss albino mice rounded out the picture. Animals receiving intratumoral injections of either the doxorubicin gel or the dual-drug gel at a 5:1 ratio survived the full 14-day observation period with normal behavior, feeding and weight gain. Hematology showed no significant changes in hemoglobin, red blood cell or white blood cell counts, biochemistry revealed normal AST, ALT, urea and creatinine levels, and histopathological examination of the breast, heart, liver, spleen and kidney found no necrosis, inflammation or degenerative changes in any treatment group. The authors are candid about the study’s limits: cytotoxicity was demonstrated only in MCF-7 cells, not in triple-negative models such as MDA-MB-231, and no reserpine-only gel group was included in the animal work, so the independent contribution of the chemosensitizer remains to be defined. Future studies, they write, will address triple-negative cell lines, orthotopic tumor models, and detailed pharmacokinetic and biodistribution profiling. Still, the convergence of computational docking, nanoscale characterization, tumor-selective release and clean safety data makes a compelling case that an old alkaloid and a smart gel, injected straight into the tumor, could give one of oncology’s workhorse drugs a second act against its own resistance mechanisms.

Subject of Research: A thermoresponsive in situ poloxamer gel co-delivering doxorubicin and reserpine to overcome multidrug resistance in breast cancer

Article Title: A novel in situ gel system for potentiating doxorubicin efficacy through reserpine co-therapy

Article References: Fiaz, H., Hameed, H., Al-Hussain, S. A., Irfan, A., Shabbir, M., & Zaki, M. E. A. (2026). A novel in situ gel system for potentiating doxorubicin efficacy through reserpine co-therapy. Journal of Saudi Chemical Society, 30(2), Article 24. https://doi.org/10.1007/s44442-026-00071-z

Image Credits: AI Generated

DOI: 10.1007/s44442-026-00071-z

Keywords: breast cancer, doxorubicin, reserpine, in situ gel, P-glycoprotein, CD44, multidrug resistance, poloxamer, drug delivery, molecular docking, thermoresponsive hydrogel, chemosensitizer