The central innovation of the work lies in the fabrication of what the author calls CPT@PLA micronanofibers, a composite mat in which camptothecin is embedded within a matrix of poly(lactic acid), or PLA, a biodegradable and biocompatible polymer that is approved by the US Food and Drug Administration and occurs naturally in the human body. For the first time, a solution containing both camptothecin and PLA was subjected to electrospinning, a technique in which a high voltage of 18 kilovolts is applied to a polymer solution flowing through a stainless-steel needle at a rate of 0.5 milliliters per hour. As the charged jet accelerates toward a grounded collector positioned 20 centimeters away, the solvent evaporates almost instantaneously, leaving behind ultrafine fibers with diameters spanning the nano and micro scales. The resulting mats were vacuum dried for 24 hours at 40 degrees Celsius to remove residual solvent and then stored at 4 degrees Celsius away from direct sunlight until analysis.

A battery of physicochemical characterization techniques confirmed that the drug had been successfully integrated into the fibrous scaffold. Scanning electron microscopy revealed that pristine PLA fibers were smooth, bead-free, and randomly organized, with average diameters of roughly 1 to 1.5 micrometers. Once camptothecin was incorporated, the fibers displayed a broader diameter distribution encompassing both nano and micro dimensions, a shift the author attributes to chemical interactions between the drug and the polymer solution that may have altered the ionic balance of the spinning jet and promoted shrinkage of the fiber diameter. Confocal laser scanning microscopy provided a particularly striking visual confirmation: because camptothecin is intrinsically fluorescent, the fibers glowed brightly under laser excitation at 488 and 543 nanometers, demonstrating that the drug molecules were well dispersed throughout the electrospun matrix rather than clumped into aggregates.

X-ray diffraction analysis added another layer of evidence. Pure camptothecin is crystalline, exhibiting characteristic diffraction peaks at Bragg angles of 16.97 and 24.8 degrees along with several minor reflections, while the PLA polymer is amorphous and shows no prominent peaks. The composite CPT@PLA fibers produced an X-ray diffraction spectrum essentially identical to that of PLA, with only a few low-intensity minor peaks remaining. This disappearance of the drug’s crystalline signature indicates that the rapid solvent evaporation during electrospinning, enabled by the enormous surface area of the forming fibers, did not give the camptothecin molecules enough time to recrystallize, trapping them instead in an amorphous state. Electron probe microanalysis mapping further verified that carbon, oxygen, and nitrogen, the key elemental components of the drug, were uniformly distributed across the fiber surfaces.

Thermogravimetric analysis, performed in a nitrogen atmosphere from 25 to 700 degrees Celsius at a heating rate of 10 degrees per minute, revealed a single-step degradation profile for both materials. Pristine PLA fibers began decomposing at around 210 degrees Celsius and lost approximately 99 percent of their mass, whereas the CPT@PLA composite fibers showed an initial degradation temperature of 280 degrees Celsius and lost about 95 percent of their mass between 200 and 400 degrees Celsius. The thermal decomposition temperature of the composite was therefore elevated by 25 percent relative to pure PLA, a shift the study attributes to the higher breakdown temperature of the encapsulated camptothecin. The first derivative curves of the thermograms showed an additional peak in the composite fibers corresponding to drug decomposition, providing yet more confirmation that camptothecin had been genuinely incorporated into the polymer matrix rather than merely adsorbed onto its surface.

With the material thoroughly characterized, the biological question became paramount: does the fiber-encapsulated drug retain its lethal activity, and how does it kill cells? To answer this, the study turned to C2C12 cells, a mouse myoblast cell line that has a long history as a model for mechanistic toxicology. These cells carry receptors that respond to diverse chemotherapeutic treatments, including agents that interfere with DNA, block RNA synthesis, and inhibit topoisomerase enzymes, and they are known to be sensitive to many apoptotic chemical stimuli. Previous researchers have used C2C12 cells to probe the anticancer properties of copper and gold compounds, making them a well-validated platform for the present investigation. Cells were seeded into 96-well plates at a density of 10,000 cells per well, allowed to adhere overnight until reaching approximately 40 percent confluence, and then exposed to either pristine PLA fibers or CPT@PLA fibers at doses of 5 and 10 micrograms per well for periods of 12, 24, and 36 hours.

The results of the Cell Counting Kit-8 assay, which quantifies cell viability by measuring absorbance of a formazan product at 450 nanometers, were unambiguous. Pristine PLA fibers showed no significant cytotoxicity across all time points, with only an insignificant drop in viability after 36 hours, confirming that the polymer itself is benign. In stark contrast, the CPT@PLA micronanofibers suppressed C2C12 cell survival dramatically, achieving approximately 73 percent inhibition at the 5 microgram dose and 80 percent inhibition at the 10 microgram dose after 36 hours of exposure. The toxicity was both time dependent and concentration dependent, and the author attributes the pronounced effect to the enhanced stability of camptothecin when sequestered within the PLA matrix, which shields the drug’s active lactone ring from hydrolysis. That ring is crucial for the spontaneous diffusion of the drug into unhealthy cells, and its preservation during electrospinning represents one of the study’s most significant technical achievements.

Phase contrast microscopy captured the morphological consequences of this cytotoxic assault. Untreated control cells displayed an organized growth pattern typical of healthy myoblasts, while cells exposed to the CPT@PLA fibers exhibited substantial disfigurement characterized by cell shrinkage and fragmentation, hallmarks of impaired cellular integrity and incipient programmed death. To identify the molecular machinery driving this demise, the study employed reverse transcriptase PCR to measure the messenger RNA expression of two key apoptotic executioners, caspase-3 and caspase-7, with expression levels normalized to the reference gene GAPDH. Exposure to the CPT@PLA micronanofibers substantially amplified the expression of both caspase genes compared with untreated controls, and the increase was dose dependent, with higher fiber concentrations producing greater expression levels.

These molecular findings fit neatly into the established mechanism of camptothecin action. By inhibiting topoisomerase I, the drug induces DNA damage that activates the intrinsic, or mitochondrial, apoptotic pathway. This triggers the release of cytochrome c from mitochondria into the cytosol, where it initiates the caspase cascade. Caspases-3 and 7 serve as the principal executors of apoptosis, dismantling cellular structures, fragmenting DNA, and ultimately dismantling the cell from within. The sensitivity of cells to camptothecin depends in part on their capacity to activate these caspases effectively, and understanding this interplay opens the door to combination therapies that could enhance caspase activation or counteract resistance mechanisms in tumors. The study also notes that camptothecin triggers cell cycle checkpoints that arrest cells in S phase, and if the resulting DNA damage proves irreparable, the cells proceed inexorably to programmed death.

The author is candid about the limitations of the current work and the road ahead. Only caspases-3 and 7 were profiled, and future studies should extend the analysis to caspase-9, the initiator caspase of the mitochondrial pathway, to complete the mechanistic picture. Detailed release kinetics of camptothecin from the fibers remain to be determined, and in vitro toxicity testing against a panel of actual cancer cell lines, including breast, ovarian, colon, lung, and stomach cancers, is strongly advocated. Nevertheless, the study establishes an important proof of principle: electrospinning can encapsulate a fragile plant-derived prodrug in a biocompatible polymer matrix without destroying its active lactone ring, while simultaneously improving its thermal stability, protecting it from hydrolysis, and preserving its capacity to kill cells through the caspase-mediated apoptotic pathway. As electrospun nanofibers continue to find applications as bandages, implants, and drug delivery scaffolds, this work suggests that the humble fiber mat may yet become a formidable weapon in the ongoing war against cancer.

Subject of Research: Camptothecin-loaded PLA micronanofibers inducing caspase-mediated apoptosis in C2C12 cells

Article Title: Caspase mediated apoptosis induced by CPT@PLA micro-nanofibers in C2C12 cells: insights into probable mechanism

Article References: Amna, T. (2025). Caspase mediated apoptosis induced by CPT@PLA micro-nanofibers in C2C12 cells: insights into probable mechanism. Discover Toxicology, 2(1), Article 12. https://doi.org/10.1007/s44339-025-00024-y

Image Credits: AI Generated

DOI: 10.1007/s44339-025-00024-y

Keywords: camptothecin, electrospinning, poly(lactic acid), micronanofibers, apoptosis, caspase-3, caspase-7, C2C12 cells, drug delivery, topoisomerase I, nanomedicine, cytotoxicity