Polymeric PLGA Nanoparticles Loaded with Acalypha monostachya Leaf Hexane Extract: A Novel Strategy for Antineoplastic Activity

Abstract

Background/Objectives: Acalypha monostachya is used in rural communities in Mexico as a traditional remedy for cancer, and we previously observed cytotoxic activity of its extracts against MDA-MB-231 and HeLa cells. Methods: Because lipophilic plant fractions disperse poorly in water, we encapsulated the hexane leaf extract (LHE) of A. monostachya in poly (lactic-co-glycolic acid) (PLGA) nanoparticles prepared by nanoprecipitation, characterized them physicochemically, and evaluated their in vitro cytotoxicity. Results: The selected extract/polymer ratio (5/50, w/w) produced nanoparticles with a mean diameter of 131.4 ± 0.5 nm and a PDI of 0.122 ± 0.028, with an encapsulation efficiency of 92.03% and a loading of 8.43%. We next evaluated cytotoxicity by MTT after 24 h in HeLa and MDA-MB-231 cells and compared the response with non-tumorigenic HaCaT keratinocytes. Encapsulation increased potency relative to free LHE, yielding IC₅₀ values of 30 µg/mL (HeLa), 60 µg/mL (MDA-MB-231), and 95 µg/mL (HaCaT). These values corresponded to selectivity indices of 3.2 (HaCaT/HeLa) and 1.6 (HaCaT/MDA-MB-231). Conclusions: Overall, encapsulation of LHE in PLGA nanoparticles yields an aqueous PLGA nanoparticle suspension and is associated with improved in vitro potency while maintaining measurable selectivity against cancer cells.

Keywords: Acalypha monostachya; cancer; cytotoxicity; nanomedicine; nanoparticles; tumor cell lines.

Figure 1

Evaluation of relative cell viability by MTT assay after treatment with HE of A. monostachya leaves (LHE), stems (SHE), and flowers (IHE) in immortalized HaCaT human epithelial cells (blue), HeLa cervical cancer cells (red), and MDA-MB-231 triple-negative breast cancer cells (green). Data are expressed as relative viability (%) compared with the untreated control (mean ± SD, n = 6). One-way ANOVA with Tukey’s post hoc test was used to compare differences between groups (* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001).

Figure 2

Illustrative chromatograms from UHPLC-UV-MS analysis of the LHE of A. monostachya (10 mg/mL). (A) UV chromatogram at 254 nm, (B) base peak chromatogram under positive ESI conditions, and (C) base peak chromatogram under negative ESI conditions.

Figure 3

Hemolytic activity of the LHE of A. monostachya at different concentrations (0–500 μg/mL) on human erythrocytes. Data are presented as mean ± SD (n = 3). Statistical analysis was performed by one-way ANOVA with Dunnett’s test (p ≤ 0.05). * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 4

Cytotoxicity analysis by MTT assay in cells treated for 24 h with different concentrations of NPs. Relative cell viability percentages are shown for HaCaT and HeLa cell lines. Both cell lines exposed to concentrations of 0, 10, 50, 100, 300, and 500 µg/mL of NPs are shown. Arrows point out that HeLa cancer cells are affected by a lower concentration than immortalized HaCaT cells, especially with the 5/50 LHE-PLGA NP formulation. n = 8. * p ≤ 0.05, Tukey’s multiple comparison test vs. HaCaT cells.

Figure 5

Raman spectra of empty PLGA NPs, LHE encapsulated into PLGA NPs (LHE-PLGA-NPs), and unencapsulated LHE of A. monostachya.

Figure 6

Fourier-transform infrared spectra of empty PLGA NPs, LHE of A. monostachya, and LHE-PLGA NPs.

Figure 7

Scanning electron microscopy (SEM) images of empty PLGA NPs (a) and NPs loaded with LHE of A. monostachya (b). Magnified images of representative regions are shown (bottom panel). Scale bar: 200 nm. (c) Histogram showing the particle size distribution of PLGA NPs. (d) Histogram showing the particle size distribution of PLGA NPs loaded with LHE, indicating a slight increase in the average particle size after LHE incorporation.

Figure 8

Stability evaluation of unloaded PLGA NPs and those loaded with LHE of A. monostachya (LHE-PLGA NPs), stored at 4 °C and 25 °C for 3 months. The following were analyzed: average particle size (top left), polydispersity index (PDI) (top right), and zeta potential (mV) (bottom). Data represent mean ± standard deviation (n = 3).

Figure 9

Effect of the different treatments in HaCaT, HeLa, and MDA-MB-231 cells. Vehicle control cells exhibited an elongated, fibroblast-like morphology with minimal cytoplasmic granulation, consistent with healthy cell appearance. Cells were treated with 10 µg/mL of extract for 24 h. Morphological alterations (magnification) such as rounding and decreased cell confluence are observed. Scale bar: 300 µm.

Figure 10

Effect of the different treatments in HaCaT, HeLa, and MDA-MB-231 cells. Vehicle control cells exhibited an elongated, fibroblast-like morphology with minimal cytoplasmic granulation, consistent with healthy cell appearance. Cells were treated with 50 µg/mL of extract for 24 h. Morphological alterations (magnification) such as rounding and decreased cell confluence are observed. Scale bar: 300 µm.

Figure 11

Effect of the different treatments in HaCaT, HeLa, and MDA-MB-231 cells. Vehicle control cells exhibited an elongated, fibroblast-like morphology with minimal cytoplasmic granulation, consistent with healthy cell appearance. Cells were treated with 100 µg/mL of extract for 24 h. Morphological alterations (magnification) such as rounding and decreased cell confluence are observed. Scale bar: 300 µm.

Figure 12

Evaluation of relative cell viability by MTT assay after treatment with LHE of A. monostachya, encapsulated LHE-PLGA NPs, and empty PLGA NPs. Data are expressed as a percentage of relative viability relative to the untreated control (mean ± SD, n = 6). One-way ANOVA with Tukey’s post hoc test was used to compare differences between groups (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Figure 13

Analysis of apoptosis and necrosis induced by different treatments in HaCaT, HeLa, and MDA-MB-231 cells. Representative fluorescence microscopy images showing red fluorescence (necrosis), green fluorescence (apoptosis), and merged channels after treatment with doxorubicin, unencapsulated HE (100 µg), and HE-PLGA NPs (100 µg). Doxorubicin predominantly induced necrosis, whereas HE-PLGA NPs mainly promoted apoptotic cell death in cancer cells, with limited cytotoxic effects on HaCaT cells. (Magnification: 40×).