Evaluation on the manufacturing, properties, and potential use of Chara vulgaris extract cubosome nanoformulation to mitigate hepatic and renal damage from Solid Ehrlich carcinoma: in vitro and in vivo investigation

Maha Alsunbul; Thanaa A El-Masry; Enas I El Zahaby; Mostafa M El-Sheekh; Mohamed M S Gaballa; Abdullah A Saber; Eman E Elmohamady; Heba Kamel Mahmoud Badawy; Abeer Saad Gawish; Amal Abdullah Alrashidi; Reem ALQahtani; Maysa M F El-Nagar

doi:10.1186/s11671-026-04527-4

. 2026 Apr 14;21(1):120. doi: 10.1186/s11671-026-04527-4

Evaluation on the manufacturing, properties, and potential use of Chara vulgaris extract cubosome nanoformulation to mitigate hepatic and renal damage from Solid Ehrlich carcinoma: in vitro and in vivo investigation

Maha Alsunbul ¹, Thanaa A El-Masry ^2,³, Enas I El Zahaby ⁴, Mostafa M El-Sheekh ⁵, Mohamed M S Gaballa ⁶, Abdullah A Saber ⁷, Eman E Elmohamady ⁸, Heba Kamel Mahmoud Badawy ⁹, Abeer Saad Gawish ¹⁰, Amal Abdullah Alrashidi ¹, Reem ALQahtani ¹, Maysa M F El-Nagar ^11,^✉

PMCID: PMC13079261 PMID: 41981339

Abstract

The family Characeae contains a variety of bioactive substances with structurally diverse chemical compositions and biological functions. This study aimed to evaluate the potential role of Chara vulgaris methanolic extract cubosome nanoformulation in the treatment of the hepatic and renal damages caused by solid Ehrlich carcinoma (SEC). C. vulgaris extract was characterized using gas chromatography-mass spectroscopy (GC–MS), and the biosynthesized C. vulgaris nanoformulation was identified using the fundamental analytical approaches. Female Swiss albino mice were used to examine the effect of C. vulgaris nanoformulation against SEC-induced hepatic and renal damage, which were allocated randomly to eight equal groups (n = 6). GC–MS uncovered the presence of several bioactive compounds in the C. vulgaris methanolic extract, particularly phytol, oleic, phthalic, and palmitic acids, as well as hexadecanoic acid, 2,3-dihydroxypropyl ester, and 9-octadecenoic acid, 1,2,3-propanediol ester, (E, E, E)-, which may play a key biological functional defense role. All the evaluated liver (AST, ALT, ALP, and total bilirubin) and kidney (urea and creatinine) functions were markedly decreased in the in vivo research as a result of the C. vulgaris nanoformulation treatment. The histopathological examination of the liver and kidney supports our observations well. Moreover, the immunohistochemical markers (ER-α, Ki-67, PCNA, and VEGF) were significantly downregulated in the C. vulagris nanoformulation treatment, while caspase-3 was upregulated as compared to the untreated SEC mice. Our findings suggest that C. vulgaris nanoformulation is a potential formulation strategy for mitigating the liver and kidney damage caused by SEC.

Graphical abstract

Keywords: Chara vulgaris, Cancer treatment, Liver and kidney damage, Nanoparticles, Solid Ehrlich carcinoma

Introduction

Breast cancer’s high prevalence of morbidity and death makes it a worldwide health concern for women. Even with adjuvant treatment, the five-year survival rate for metastatic breast cancer is less than 30% [1]. The International Agency for Research on Cancer (IARC) stated that 2.3 million new cases (11.7%) and a mortality rate of 6.9% in 185 countries [2]. Metastatic breast cancer is inoperable, and it is the leading cause of most fatalities from breast cancer. Hepatic complications related to breast cancer are frequent and are usually due to metastasis of the tumor to the liver, besides the adverse effects of the chemotherapeutic agents [3]. The common clinical manifestations of hepatic metastasis of breast cancer are abdominal tenderness, anorexia, jaundice, ascites, hepatomegaly, and back pain. Extensive carcinomatous infiltration of hepatic sinusoids results in carcinomatous cirrhosis and subsequent fulminant hepatic failure [4, 5].

Moreover, one of the most common complications among patients with breast cancer is renal failure due to variable causes like chemotherapeutic agents used in breast cancer, radiotherapy-induced nephritis or thrombotic microangiopathy, renal metastasis, or paraneoplastic hypercalcemia [6] Cases of metastatic breast cancer are usually inoperable, so chemotherapy is the cornerstone in the treatment of these cases as antimetabolites e.g. methotrexate and doxorubicin, monoclonal antibodies e.g. trastuzumab, DNA alkylating agents e.g. cisplatin, antimitotic e.g. paclitaxel. Hormonal therapy, which includes aromatase inhibitors like letrozole and estrogen receptor modulators like tamoxifen, is also essential in treating such conditions [7]. These chemotherapeutic agents have multiple adverse effects, so research is being done to find new, effective drugs with fewer adverse effects. Ehrlich tumors fall within the category of spontaneous adenocarcinomas in mice. Because they are a transplantable tumor model, it is now easy to investigate the anticancer effects of different substances [8].

Natural compounds and their derivatives are an excellent starting point for the search for novel small-molecule cancer therapy agents [9]. Recently, a number of screening studies have been carried out to find novel cytotoxic biochemical components from microalgae, particularly cyanobacteria and green algae [10]. It has been reported that algae are characterized by the presence of several promising antibacterial, anthelminthic, antiviral, antitumoral, anti-inflammatory, and anticoagulant bioactive constituents [11]. The streptophyte Chara vulgaris (C. vulgaris) is a cosmopolitan green macroalga that can grow in all kinds of fresh to brackish permanent, periodic, and ephemeral waters. Besides its uses in food, pharmaceutical, and cosmetic industries, it has some therapeutic characteristics [12]. Previous studies pinpointed that some members of the family Characeae, such as C. globularis, C. elegans (currently regarded as a synonym of C. zeylanica var. elegans), and C. baltica, exhibit cytotoxic and antineoplastic activities [13, 14].

Devices known as drug delivery systems move a medicinal substance to a certain location within the body. Findings suggest that the Nano drug delivery system (NDDS) is a viable choice for treating serious illnesses. Because of this, a variety of carriers have been created during the last ten years, and the development of additional ones is happening at a staggering rate [15]. Recently, cubosomes (Cub) have emerged as a new member of the nanocarrier family, serving as a non-lamellar equivalent to liposomes. Cubosomes, like liposomes, are particles made out of lipid bilayers that self-assemble in an aqueous solution. Three-dimensional patterns with continuous hydrophobic and hydrophilic patches are produced by the twisting and deformation of the lipid bilayers in cub. They have a thermodynamically stable three-dimensional structure that resembles a honeycomb (100–500 nm) [16]. Cub’s liquid-crystalline structure, which may enable continuous drug release over extended periods, is its main benefit over liposomes [17]. Because the particle’s increased lipid content provides a bigger surface area, Cub can provide more encapsulation than other drug delivery techniques [18].

Cub is formulated with biodegradable, biocompatible, and bio-adhesive lipids, which can be considered an advantage over dendrimers, which may cause toxicity due to charges and the nature of the building blocks [19]. Poloxamer 407 is a block copolymer that contains hydrophilic ethylene oxide (EO) and hydrophobic propylene oxide (PO) units. It displays surfactant properties. They may also interact with hydrophobic particles and biological membranes. Over the past ten years, several assessments on the use of poloxamer 407 for drug delivery have been published. By appropriately modifying hydrophobic nanoparticles to assist them in evading phagocytosis, sustainable circulation can be achieved [20].

In this study, C. vulgaris species was selected for its unique phytochemical profile, especially for its richness in polyphenolic and flavonoid compounds, which are recognized for their antioxidant and biological properties. In fact, previous studies showed the potential of C. vulgaris for biomedical applications, considering its antioxidant, antimicrobial, and anticancer properties. In addition, C. vulgaris is a widely distributed and easily collected species, which can be considered an environmentally friendly option when compared to other algae and plant extracts [21–23].

Significantly, recent developments in algal-based nanoformulations have been largely dependent on a small number of well-characterized species of algae, such as Spirulina sp, Chlorella sp, and various brown algae, with most of the scientific studies being conducted with a focus on their use as reducing agents, stabilizers, or drug carriers for conventional drugs [24–31]. To the best of our knowledge, this is the first report that uses a C. vulgaris-based cabsomal nanoformulation with a newly designed formulation strategy that has never been reported before in the literature. It is important to note that our approach provides an overall framework that includes original formulation design, physicochemical characterization of the nanoformulation, and biological evaluation of the nanoformulation. In this regard, it is clear that the present study fills an important gap in the literature by providing an innovative candidate of algal species and a biological evaluation of the nanoformulation.

Therefore, this study aimed to evaluate the C. vulgaris methanolic extract and its cubosomal nanoformulation to alleviate hepatic and renal damage caused by Solid Ehrlich Carcinoma (SEC).

Materials and methods

Drugs and chemicals

Poloxamer 407, Glycerol α monooleate, ethyl alcohol (highly analytical grade), and deionized water were brought from BASF Corporation (USA), TCI (Japan), Merck (Darmstadt, Germany), and Stakpure Waters (Milford, MA, USA). Also, Tamoxifen was provided by the Egyptian company Techno Pharmaceutical Co. (Alexandria, Egypt).

Collection of C. vulgaris biomass

A population of C. vulgaris was sampled on 4th June 2022 from a freshwater pool (24° 33′ 24.6″ N, 30° 38′ 59.1″ E) in the Baris Oasis, the Egyptian Western Desert. The specimens were morphologically identified, and also genetically (unpublished data, M. El-Sheekh & A.A. Saber), following the relevant taxonomic literature [32–34].

In vitro study

Preparation of C. vulgaris extract

After mixing 100 ml of 80% methanol with 10 g of C. vulgaris, the mixture was shaken (VS-8480) [23, 35] for 72 h at 25 °C. The ethanolic extract (Sigma 2-16KL Centrifuge, Osterode, Germany) was obtained by centrifuging for 15 min at 4000 rpm. The collected supernatant was then concentrated using a rotary vacuum evaporator, and it was then freeze-dried to get the final methanolic extract of Chara vulgaris. It was kept out of direct sunlight and at 4 °C until it was utilized.

Methanolic extract of C. vulgaris analyzed using GC-MS

C. vulgaris methanolic extract (10 mg diluted in 1.5 ml methanol (80%) with 3 µl injection volumes) was subjected to a chemical composition analysis using GC/MS equipment (Thermo Scientific, Lenexa, USA, Trace GC-ISQ mass spectrometer). The temperature program was designed to increase the temperature by 10 degrees Celsius every minute, from 50 to 280 degrees. The injector was 220 ◦C, the interface was 220 °C, and the source was 200 °C. The carrier gas was helium at a rate of one milliliter per minute. By comparing the mass spectra and component retention periods with mass spectral databases, namely NIST 05 (NIST/EPA/NIH mass spectral library) [36].

Preparation of nanoparticles

Cubs were synthesized utilizing the simple emulsification of glycerol monooleate with a poloxamer 407, followed by sonication and homogenization [16]. In brief, 500 mg of GMO and 100 mg of Poloxamer 407 were mixed and allowed to melt with the aid of a hot plate magnetic stirrer (Stuart, Caliber Scientific USA) at 70 °C. 10 ml of alcoholic extract of C. vulgaris (equivalent to 12 mg dry powder) was transferred into the Poloxamer 407/glycerol monoleate mixture with continuous stirring for an additional ten minutes. Deionized water (10 ml at 70 °C) was added gradually to the previous mixture with continued stirring at 400 rpm for an additional 10 min. The colloidal dispersion was further subjected to sonication utilizing a probe sonicator (Sonic Vibra Cell, USA) for 5 min in an ice bath (10 s pulse and 10 s pause) and amplitude of 88% (power is 130 W), followed by homogenization for 10 min at 10,000 rpm.

Characterization of Cub-NPs–C. vulgaris

UV spectrum of Cub-NPs–C. vulgaris

A UV-visible spectrophotometer (Shimadzu UV-VIS spectrophotometer, UV-1900I, Japan) was utilized to investigate the absorption spectra of both the methanolic lyophilized powder of C. vulgaris diluted in ethyl alcohol and Cub-NPs– C. vulgaris. The samples were scanned in the spectral range of 190–900 nm. A quartz cuvette with a maximum volume of 3 mL was used for spectrum scanning. The path length of light inside the cuvette was 1 cm. Experiments were repeated in triplicate (n = 3), with mean results and standard deviations given for each [37].

Drug content and % entrapment efficiency (%EE) of Cub-NPs–C. vulgaris

The average drug content was determined for Cub-NPs–C. vulgaris. An accurately measured volume (300 µl) of Cub-NPs–C. vulgaris was relocated into a 10 ml volumetric flask, the volume was completed with absolute alcohol, serial dilution was conducted, and the absorbance at 222 nm was measured, and the corresponding concentration was determined utilizing a standard curve with the linearity of 100–900 µg/ml.

The % entrapment efficiency %EE was calculated according to the following equation:

Particle size, zeta potential, polydispersity index (PDI)

Using dynamic light scattering (DLS) and a Zetasizer (Malvern Instruments, Malvern, UK), the mean particle diameter (MD), polydispersity index (PDI), and Zeta potential of Cub-NPs–C. vulgaris were ascertained. Before being measured, each sample was diluted with deionized water to reach a light scattering intensity of about 300 Hz. The samples were then measured three times at 25 °C (allowing them to equilibrate for five minutes) and analyzed three times each [38].

SEM and TEM

The SEM method was used to ascertain the surface properties and structure of Cub-NPs–C. vulgaris. Following thorough drying, the powder was moved to the top of the copper stub on a silicon electro-conductive chip using the colloidal dispersion that had been put on a glass slide. After a minute of gold coating, the sample was viewed by a field emission scanning electron microscope (JEOL, JSM-6510LV, Japan) at various magnifications (10 kV electron acceleration).

A transmission electron microscope (JEM1400, JEOL Ltd., Tokyo, Japan) was used to examine the Cub-NPs–C. vulgaris’s actual shape and size. Generally, the filter paper was used to deposit the Cub-NPs–C. vulgaris’s colloidal dispersion onto a grid surface covered with a membrane. They were then negatively stained with uranyl acetate dye for 3 min and dried at room temperature before being loaded into the microscope.

FTIR investigation

The functional groups were detected using infrared spectroscopy (FTIR; Perkin Elmer Fourier transform spectrometer, Bruker, USA), and an air-cooled DTGs (deuterated triglycine sulfate) detector. Concisely, a mixture including chara vulgaris, Cub-NPs–C. vulgaris, and poloxamer 407 was compacted with KBr into a disc with a hydraulic press. The disc was monitored at wavelengths ranging from − 4000 to − 400 cm − 1.

In vivo study

Animals

Female Swiss albino mice weighing between 22 and 25 g were acquired from the National Institute of Ophthalmology, Giza, Egypt. The animals were given regular food and free access to water. The research ethics committee of Tanta University’s Faculty of Pharmacy approved laboratory animal handling practices that adhered to the updated Animals (Scientific Procedures) Act 1986 in the UK and Directive 2010/63/EU in Europe (Code of Protocol (TP/RE/4/23p-0019)).

Solid Ehrlich tumor induction

The Ehrlich ascites carcinoma (EAC) cell line was supplied by the Pharmacology and Experimental Oncology Unit of the National Cancer Institute (NCI), Cairo University, Egypt. In brief, 0.2 ml of live EAC cells (1 × 10⁶ cells) were subcutaneously injected into the lower limb for the SEC model induction in Female Swiss albino mice. On the 12th day following the EAC cell injection, a 21-day course of therapy involving various medications was initiated [39].

Design for experiment

The mice were allocated into 8 groups, each consisting of six rats, at random: (1) Control vehicle; (2) SEC group: each mouse’s lower leg thigh received a subcutaneous injection of 0.2 ml (1 × 10⁶) of live EAC cells and distilled water was given to the mice; (3) SEC + TAM: Daily doses of tamoxifen (10 mg/kg, I.P.) dissolved in distilled water were given to the mice for 21 days [40]; (4) SEC+Polymer: mice received daily oral doses of cubosome polymer; (5) SEC + C. vulgaris (20): mice received daily oral doses of C. vulgaris (20 mg/kg, P.O.) dissolved in distilled water for 21 days. (6) SEC + C. vulgaris (40): mice received daily oral doses of C. vulgaris (40 mg/kg, P.O.) dissolved in distilled water for 21 days. (7) SEC + Cub-NPs–C. vulgaris (20): mice received daily oral doses of C. vulgaris -cubosome nanoformulation (20 mg/kg, P.O.) for 21 days. (8) SEC + Cub-NPs–C. vulgaris (40): mice received daily oral doses of C. vulgaris -cubosome nanoformulation (40 mg/kg, P.O.) for 21 days. The 20 and 40 mg/kg dosages were based on previously established effective dosage ranges that were within 25–50 mg/kg [23, 31]. A preliminary dose range finding study was also conducted to assess the suitability of the dosages for inducing anticancer responses in the experimental model.

Blood and tissue samples collection

The mice were sacrificed 24 h after the last treatment on day 22. All of the mice were euthanized (isoflurane anesthesia), and the blood was collected via a cardiac puncture in clean test tubes. After allowing the blood to coagulate for 15 min at 4 °C, it was centrifuged for 15 min at 4000 rpm using a Sigma 2-16KL Osterode, Germany. To perform a biochemical analysis, a serum sample was collected and kept below − 20 °C. The serum samples were then tested for liver enzymes (Alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin levels) and kidney functions (urea and creatinine). Subsequently, the mice were cervically dislocated and killed following the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020 Edition). Furthermore, both the liver and the kidney were removed and kept at room temperature in a 10% buffered formalin solution for immunohistochemical and histological examinations. Additional samples of liver and kidney tissues were kept for biochemical analysis at − 80 °C.

Determination of liver enzyme

ALP (Cat. No. MDBEIS44-I), ALT (Cat. No. BEIS11-P), AST (Cat. No. MDBEIS46-I), and total bilirubin (Cat. No. MDBSIS04-I) activity in serum were determined in accordance with guidelines provided by the manufacturer using ELISA kits (SPINREACT, Santa Coloma Bas, Spain).

Kidney function assessment

Using kits supplied by SPINREACT, Santa Coloma Bas, Spain, and in compliance with the manufacturer’s guidelines, serum samples were examined for urea and creatinine serum, Cat No. BSIS32-I and BSIS13-I, respectively.

Histopathological analysis

Sections of liver and kidney tissues, each four micrometers thick, were meticulously positioned on glass slides. To ensure the best possible tissue preparation for the staining process, the tissue slices were deparaffinized using xylene to remove the embedding media. After deparaffinization, the sections were successively hydrated in decreasing percentages of ethanol solutions: 100% ethanol at first, then 95%, 80%, and 70%. Every concentration step lasted five minutes. The ensuing histological staining processes were made easier by the efficient substitution of water for xylene during this progressive rehydration phase. Hematoxylin and Eosin (H&E) were used to stain the liver and kidney tissue slices to evaluate the overall histological architecture and spot any abnormalities. The histological investigation was carried out utilizing a light microscope with 200× magnification [41]. Liver and kidney tissues were evaluated using a semi-quantitative histopathological scoring system adapted from established toxicologic pathology guidelines [42–44]. Lesions were graded on an ordinal scale from 0 to 4, reflecting both severity and the proportion of tissue affected, where 0 indicated absence, 1 minimal involvement (≤ 10% of tissue affected), 2 mild (10–30%), 3 moderate (30–60%), and 4 severe (> 60%). For the liver, the parameters assessed included hepatocellular degeneration and vacuolation, vascular congestion and hemorrhage, inflammatory cell infiltration, necrosis or apoptosis, and the presence of tumor cell nests. For the kidney, the parameters assessed comprised tubular degeneration and necrosis, glomerular atrophy and sclerosis, interstitial inflammatory infiltration, tubular dilation and casts, and interstitial fibrosis. Non-overlapping high-power fields (HPFs, 200×) were examined per section, and mean lesion scores were calculated for each animal. For each animal (n = 6 per group), liver and kidney sections were evaluated across 12 non-overlapping high-power fields. The sum of individual lesion scores yielded a total injury score for each organ, ranging from 0 to 20. All slides were anonymized and independently evaluated by two pathologists, with discrepancies resolved by consensus to ensure reproducibility and minimize observer bias. This approach provided a robust and standardized framework for quantifying histopathological injury, enabling direct comparison across experimental groups and correlation with biochemical and immunohistochemical findings.

Immunohistochemical examination

As directed by the immunohistochemical staining kit, prepared paraffin slices were exposed to 3% H₂O₂ to deactivate endogenous peroxidase. Serum-blocking and heat-induced antigen retrieval methods followed. ER-α (estrogen receptor alpha, Catalog: sc-53493, Santa Cruz, CA, USA) and Ki-67 (Catalog: sc-23900, Santa Cruz, CA, USA), VEGF (Vascular endothelial growth factor, Catalog: sc-53462, Santa Cruz, CA, USA), PCNA (proliferating cell nuclear antigen, Catalog: sc-53407, Santa Cruz, CA, USA), and Caspase-3 (Catalog: sc-7272, Santa Cruz), were added to the liver and kidney tissue slices overnight at 4 ◦C. DAB and hematoxylin counterstaining were carried out following the addition and half-hour incubation of a secondary antibody. A light microscope was used to examine the stained slides. For ER-α, immunopositive regions were quantified in ten randomly selected micrographs per animal using ImageJ software (Fiji Image J, NIH, Bethesda, MD, USA), expressed as the percentage of positively stained area as described by [45]. Ki-67 expression was assessed at 200× magnification by counting positively stained nuclei in ten randomly selected high-power fields, with results averaged to estimate the percentage of proliferating cells following the methods of [46, 47]. PCNA expression was evaluated under a double-blind protocol at 200× magnification across ten high-power fields, expressed as the percentage of positively stained cells in line with [48, 49]. VEGF expression was quantified using ImageJ by calculating the mean percentage of positively stained areas in liver tissues and renal glomeruli/tubules across ten randomly selected fields, consistent with the approaches of [50–51]. Finally, Caspase-3 activity was determined by analyzing ten random, non-overlapping fields per sample using ImageJ to calculate the mean surface area fraction of immunopositive cells, as reported by [52–53]. All values were expressed as mean ± SD, and detailed quantification tables are provided in the Results section to complement representative images.

Statistical analysis

The results were expressed as mean ± SD. To determine the differences between the groups, a one-way ANOVA was followed by Tukey’s multiple-comparative tests. Before ANOVA, data were assessed for normality and homogeneity of variances, and no violations of these assumptions were detected. Statistical significance was defined as p < 0.05. All statistical analyses were conducted using GraphPad Prism software (Version 10; GraphPad Software Inc., La Jolla, CA, USA).

Results

In vitro analysis

Examination of C. vulgaris methanolic extract by GC-MS

As seen in Fig. 1, a variety of bioactive compounds with a broad spectrum of biological activity were identified in the C. vulgaris methanolic extract. The bioactive characteristics of these substances are listed in Table 1, which mostly consisted of phytol (14.68%), oleic acid (13.92%), phthalic acid (13.72%), hexadecanoic acid, 2,3-dihydroxypropyl ester (9.64%), palmitic acid (4.67%), and 9-octadecenoic acid, 1,2,3-propanediol ester, (E, E, E)- (3.42%).

Table 1.

GC-MS analysis of C. vulgaris methanolic extract

RT	Compound Name	PA %	Biological activity
24.99	1-Heptatriacotanol	2.09	Antioxidant, anticancer, and anti-inflammatory activity
27.49	Hexadecanoic acid, 2,3-dihydroxypropyl ester	9.64	Antioxidant activity
27.82	Hexadecanoic acid, Methyl ester (ethyl palmitate)	4.67	Antioxidants, antidiabetic, and anti-inflammatory activities
29.51	Linoleic acid ethyl ester	2.25	Anti-inflammatory activity
29.68	10-octadecadienoic acid, methyl ester	2.29	Antioxidant, anticancer, and anti-inflammatory activity
31.01	Phytol	14.68	Anti-inflammatory, antioxidant, and antimicrobial activity
31.10	9-Octadecenoic acid,1,2,3-propanetriyl ester, (E, E,E)-	3.42	Antioxidant, anticancer, and anti-inflammatory activity
33.99	Oleic Acid	13.92	Antioxidants, antibacterial, and anti-inflammatory activities
36.71	1,2-benzene dicarboxylic acid	13.72	Anticancer and antiproliferation Activities
36.98	9,12,15-Octadecadienoic acid	2.04	Antioxidant, anticancer, and anti-inflammatory activities
38.18	Isochiapin B	2.56	Antioxidants and antibacterial activities

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RT: Retention Time; PA: Peak Area. A variety of chemicals’ biological activity was sourced from the PubChem database website (https://pubchem.ncbi.nlm.nih.gov/)

Characterization of Cub-NPs–C. vulgaris

Calibration curve and UV spectrum of Cub-NPs–C. vulgaris

The UV-visible spectroscopic spectrum was scanned from 190 to 900 nm on a UV-visible spectrophotometer to determine the presence of phytochemical substances in methanolic extract, and spectral peaks were examined by comparing the spectrum of methanolic extract with the suitably diluted sample of Cub-NPs–C. vulgaris in absolute alcohol (Fig. 2A). A standard calibration curve for methanolic lyophilized powder of C. vulgaris was prepared via serial dillution with ethanol to prepare the following concentrations: with the following concentrations: 100, 200, 600, and 900 µg/ml (Fig. 2B). Absorbance was estimated utilizing absolute alcohol as a blank; the scanning revealed the presence of characteristic peaks for phenolic bioactive compounds at λ _max 200 and 220 nm. The exhibition of flavonoid compounds showed peaks between 230 and 290, 300–360, and 234–676 nm.

Fig. 3 — SEM of Cub-NPs–*C. vulgaris* (A) and TEM of Cub-NPs–*C. vulgaris* (B)

Fig. 2 — A The UV spectrum of the methanolic extract of *C. vulgaris* and Cub-NPs–*C. vulgaris* (X axis represents wavelength (nm ) and Y axis represents the extent of UV absorbance). B Standard Calibration curve of methanolic extract of *C. vulgaris* (100–900 µg/ml, the X axis represents ethanolic extract concentration after dilution in ethanol (µg/ml) and Y axis represents the corresponding absorbance of each concentration at ʎ_max 220 nm)

Particle size, zeta potential, PDI, and drug content of Cub-NPs–C. vulgaris

Dynamic light scattering (DLS) was used to investigate the particle size of Cub-NPs–C. vulgaris; the mean particle size was 154.87 ± 1.9 nm. The average zeta potential was − 22.86 ± 1.38mV, while the mean PDI was 0.26 ± 0.03. The average drug content was 1.06 ± 0.14 mg/ml, and %EE was 88.31 ± 11.8% (Table 2).

Table 2.

Polymeric Cub-NPs–C. vulgaris characteristics

Parameter	Mean	Range
Particle size (nm)	154.87 ± 1.90	152.9–156.7
Zeta potential (mV)	− 22.86 ± 1.36	− 24.32 to − 21.38
PDI	0.26 ± 0.03	0.224–0.293
Drug content (mg/ml)	1.06 ± 0.14	0.922–1.207
%EE	88.31 ± 11.9	76.83–100.58

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Data are presented as mean ± SD, n = 3

SEM and TEM examination for Cub-NPs–C. vulgaris

SEM was used to study the structure and surface properties of Cub-NPs–C. vulgaris (magnification power 450000) (Fig. 3A). The size and actual shape of the Cub-NPs–C. vulgaris were determined using transmission electron microscopy. The TEM images illustrated the honeycomb structure of the cubosome (Fig. 3B).

FTIR analysis

The FTIR of C. vulgaris demonstrated a characteristic peak of hydroxyl group stretching at − 3500/ cm, and two peaks at-2937/cm and − 2904/cm due to the asymmetric and symmetric stretching vibration of C–H groups, respectively. A sharp peak at − 1666/cm, which stands for nitro compounds. The FTIR of poloxamer 407 showed a peak at − 1120/cm, which is related to the C–O stretch. The spectrum showed many medium peaks which can be related to C–C stretching of alkane group in the range of − 1200 to − 800/cm (Fig. 4). The FTIR of Cub-NPs–C. vulgaris showed a significant decrease in the % of transmittance of the hydroxyl group in the range − 3500 to − 3200/Cm, the two peaks representing C–H stretching almost disappeared, and the peaks at − 1666 to − 1485/cm became sharper. The characteristic C–O stretching peak of poloxamer 407 became sharper.

Fig. 4 — FTIR of Cub-NPs–*C. vulgaris*, *C. vulgaris*, ascorbic acid, and poloxamer 407

In vivo analysis

Effect of Cub-NPs–C. vulgaris on liver and kidney function biomarkers

As revealed in Table 3, SEC induced a significant rise (428.78%, 446.14%, 265.91%, and 245.09%, respectively) in the serum content of ALT, AST, ALP, and total bilirubin in comparison to a normal control group. In comparison, administration of Tam and Cub-NPs–C. vulgaris (20 and 40 mg/Kg) showed a significant reduction in ALT serum levels (13.11%, 24.86%, and 33.16%, respectively), in serum AST levels (21.11%, 31.49%, and 39.23%, respectively), in serum ALP levels (28.31%, 36.09%, and 39.53%, respectively) and serum total bilirubin levels (47.16%, 53.41%, and 59.66%, respectively) as compared with the SEC group. Furthermore, Cub-NPs–Chara vulgaris (20 and 40 mg/Kg)-treated mice displayed a noteworthy decline in serum levels of ALT, AST, ALP, and total bilirubin compared with C. vulgaris (20 and 40 mg/Kg)-treated mice (Table 3).

Table 3.

Effect of Cub-NPs–C. vulgaris on liver function biomarkers

Group	ALT (U/L)	AST (U/L)	ALP (U/L)	Total Bilirubin (mg/dl)
Normal control	37.35 ± 5.5	42.15 ± 7.48	115.93 ± 13.32	0.51 ± 0.11
SEC	197.5 ± 14.6^*	230.2 ± 12.21^*	424.2 ± 11.09^*	1.76 ± 0.13^*
SEC + TAM	171.6 ± 10.55^*a	181.6 ± 11.08^*,a	304.12 ± 11.14^*,a	0.93 ± 0.06^*,a
SEC+Polymer	196.3 ± 23.36^*	229.0 ± 9.43^{*, b}	424.0 ± 3.6^{*, b}	1.75 ± 0.12^{*, b}
SEC + C. vulgaris (20)	187.8 ± 14.76^*,c	210.6 ± 7.24^{*,b, c}	376.7 ± 8.76^{*,a, b,c}	1.21 ± 0.07^{*,a, b,c}
SEC + C. vulgaris (40)	179.30 ± 20.10^{*,b, c}	202.3 ± 17.08^{*,a, c}	324.12 ± 7.35^{*,a, c}	1.07 ± 0.06^{*,a, c}
SEC + Cub-NPs–C. vulgaris (20)	148.4 ± 9.82^{*,a, b}	157.7 ± 14.58^{*,a, b}	271.1 ± 19.73^{*,a, b}	0.82 ± 0.04^*,a
SEC + Cub-NPs–C. vulgaris (40)	132.0 ± 5.51^{*,a, b}	139.9 ± 10.8^{*,a, b}	256.5 ± 19.16^{*,a, b}	0.71 ± 0.12^{*,a, b}

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Data are presented as mean ± SD, n = 6. ^* Significant difference from the normal control group. ^a Significant difference from the SEC group. ^b Significant difference from SEC + TAM group. ^C Significant difference from Cub-NPs–C. vulgaris (40) group. TAM: Tamoxifen, C. vulgaris: Chara vulgaris, and Cub-NPs: Cubosomes nanoformulation. Significant difference accepted at p < 0.05

In the same context, Table 4 exposed that SEC induced a significant rise (231.24% and 371.43%, respectively) in the serum content of urea and creatinine, in comparison to a normal control group. In contrast, administration of Tam, Cub-NPs–C. vulgaris (20 and 40 mg/Kg) showed a significant reduction in Urea serum levels (40.33%, 56.09%, and 59.69%, respectively), and in serum creatinine levels (25.25%, 46.46%, and 61.62%, respectively) as compared with the SEC group. Moreover, Cub-NPs–C. vulgaris (20 and 40 mg/Kg)-treated mice presented a notable decline in serum levels of urea and creatinine compared with Chara vulgaris (20 and 40 mg/Kg)-treated mice (Table 4).

Table 4.

Effect of Cub-NPs–C. vulgaris on kidney function biomarkers

Group	Urea (mg/dl)	Creatinine (mg/dl)
Normal control	24.2 ± 4.12	0.42 ± 0.11
SEC	80.16 ± 10.17^*	1.98 ± 0.2^*
SEC + TAM	47.83 ± 7.44^*a	1.48 ± 0.07^*,a
SEC+Polymer	79.98 ± 4.29^{*, b}	1.96 ± 0.08^{*, b}
SEC + C. vulgaris (20)	70.54 ± 8.34^{*,b, c}	1.63 ± 0.08^{*,a, c}
SEC + C. vulgaris (40)	64.50 ± 3.21^{*,a, b,c}	1.37 ± 0.03^{*,a, c}
SEC + Cub-NPs–C. vulgaris (20)	35.2 ± 1.97^{a, b}	1.06 ± 0.11^{*,a, b,c}
SEC + Cub-NPs–C. vulgaris (40)	32.31 ± 2.82^{a, b}	0.76 ± 0.05^{*,a, b}

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Histopathological analysis

Histopathological investigation of tumor tissue across different treatment groups was assessed in Fig. 5. In the Normal control group, both liver and kidney tissues maintain normal architecture and cellular structure. The liver shows well-organized hepatocytes arranged in regular cords, with hexagonal cells, centrally located nuclei, and intact central veins and portal triads, free from inflammation, fibrosis, or degeneration (Fig. 5A1). Similarly, the kidney demonstrates healthy glomeruli and tubules with no signs of degeneration or inflammatory infiltration (Fig. 5B1).

In the SEC group, there is severe disruption in both liver and kidney structures. The liver exhibits significant architectural disorganization, with large nests of tumor cells demonstrating nuclear pleomorphism, abundant mitotic activity, vacuolar degeneration, and severe congestion, accompanied by an inflammatory cell presence (Fig. 5A2). In the kidneys, there is extensive tubular degeneration, glomerular atrophy, localized tubular necrosis, moderate inflammatory cell infiltration, and occasional tumor cells (Fig. 5B2).

In contrast, the SEC + TAM group showed moderate structural improvement in both organs. The liver exhibits reduced tumor cell density, occasional apoptotic bodies, and decreased mitotic activity, indicating a partial response to treatment, with some tumor nests still present (Fig. 5A3). In the kidney, there are mild signs of glomerular recovery and improved tubular morphology, though moderate cell infiltration remains (Fig. 5B3).

In the SEC+Polymer group, the histopathological improvement is very mild. The liver continues to show predominant tumor nests, with only occasional and trivial reductions in their size, and the overall architecture is barely improved (Fig. 5A4). Similarly, the kidney demonstrates only isolated reductions in tubular atrophy and a slight, sporadic decrease in inflammatory infiltration, with most degenerative changes persisting (Fig. 5B4).

The SEC + C. vulgaris (20) group demonstrated moderate recovery in both the liver and kidney. The liver shows decreased tumor cell infiltration, smaller tumor clusters, and mild congestion (Fig. 5A5). Kidney tissues display mild lymphocytic infiltration, tubular dilation, and moderate improvement in glomerular and tubular structures (Fig. 5B5). Also, in the SEC + C. vulgaris (40) group, both organs show significant improvement. The liver architecture shows near-complete tumor regression, healthy hepatocytes, and no congestion (Fig. 5A6). The kidney shows substantial recovery of tubular and glomerular structures with minimal inflammatory presence (Fig. 5B6).

In the same manner, the SEC + Cub-NPs–C. vulgaris (20) group exhibited marked tissue recovery in both the liver and kidney. The liver shows reduced tumor nests, improved hepatocyte morphology, minimal degeneration, and increased cellular proliferation and regeneration (Fig. 5A7). Kidney structures are notably restored, with enhanced tubular cell regeneration and no signs of inflammation or fibrosis (Fig. 5B7). Furthermore, in the SEC + Cub-NPs–C. vulgaris (40) group, near-complete restoration is observed in both liver and kidney tissues. The liver has minimal degeneration, absence of tumor cells, and well-organized hepatocytes (Fig. 5A8). The kidney displays normal glomeruli and tubules with no cell infiltration or inflammation, indicating almost full recovery (Fig. 5B8).

Semiquantitative scoring of liver and kidney sections revealed marked differences among experimental groups (Table 5). In the SEC group, both organs exhibited the highest injury scores, characterized by extensive hepatocellular degeneration, necrosis, vascular congestion, inflammatory infiltration, and prominent tumor nests in the liver, alongside severe tubular degeneration, glomerular atrophy, interstitial inflammation, and fibrosis in the kidney. These findings were consistent across animals, yielding mean total scores of 15.0 ± 1.2 for the liver and 14.3 ± 1.03 for the kidney. In the SEC + TAM group, Tam treatment partially ameliorated these lesions, reducing mean scores to 8.8 ± 0.9 (liver) and 8.2 ± 0.8 (kidney). Histologically, this corresponded to attenuated necrosis and inflammatory infiltration, though residual degenerative changes and occasional tumor nests persisted.

Table 5.

Histopathological scoring of liver and kidney injury

Group	Liver	Kidney
Normal control	2.0 ± 0.8	2.5 ± 0.6
SEC	15.0 ± 1.2	14.3 ± 1.03
SEC + TAM	8.8 ± 0.9	8.2 ± 0.8
SEC+Polymer	14.0 ± 1.02	14.0 ± 1.01
SEC + C. vulgaris (20)	8.3 ± 0.9	7.7 ± 0.9
SEC + C. vulgaris (40)	5.5 ± 1.3	4.5 ± 0.8
SEC + Cub-NPs–C. vulgaris (20)	4.0 ± 0.08	2.7 ± 0.05
SEC + Cub-NPs–C. vulgaris (40)	3.0 ± 0.07	2.5 ± 0.05

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Data are presented as mean ± SD, n = 12. SEC: Solid Erlish carcinoma, TAM: Tamoxifen, C. vulgaris: Chara vulgaris, and Cub-NPs: Cubosomes nanoformulation

Administration of C. vulgaris extract produced a clear dose-dependent protective effect. At 20 mg/kg, mean scores decreased to 8.3 ± 0.9 (liver) and 7.7 ± 0.9 (kidney), with reduced necrosis and milder inflammatory infiltrates. At 40 mg/kg, scores further declined to 5.5 ± 1.3 and 4.5 ± 0.8, respectively, with near-normal architecture and only focal residual lesions. The SEC + Cub-NPs–C. vulgaris group demonstrated the most pronounced protection. At 20 mg/kg, liver and kidney scores were 4.0 ± 0.08 and 2.7 ± 0.05, respectively, while at 40 mg/kg, scores approached baseline (3.0 ± 0.07 and 2.5 ± 0.05), with histology indistinguishable from normal controls in several animals. Notably, tumor nests were virtually absent, and parenchymal integrity was preserved.

Immunohistochemical examination

The expression levels of protein markers (ER-α, Ki-67, PCNA, VEGF, and caspase-3) were revealed by the immunohistochemistry investigation in different treatment groups.

The analysis of ER-α expression levels in liver and kidney tissues revealed distinct variations across experimental groups. The Normal Control group exhibited minimal ER-α expression (2.46% in the liver (Fig. 6A1) and 1.20% in the kidney (Fig. 6B1)). In contrast, the SEC group showed markedly elevated ER-α levels (35.04% in the liver (Fig. 6A2) and 17.87% in the kidney (Fig. 6B2)), reflecting tumor-induced receptor upregulation.TAM treatment significantly reduced ER-α expression (13.95% in the liver (Fig. 6A3) and 5.59% in the kidney (Fig. 6B3)), demonstrating its anti-estrogenic efficacy. The SEC+Polymer group exhibited only marginal reductions in ER-α levels (32.12% in the liver (Fig. 6A4) and 16.04% in the kidney (Fig. 6B4)), which were not statistically distinct from the SEC group, indicating a negligible therapeutic impact.

The SEC + C. vulgaris (20) group treatment yielded liver and kidney averages of 18.30% (Fig. 6A5) and 7.25% (Fig. 6B5), respectively. SEC + C. vulgaris (40) group showed slightly lower levels, with averages of 16.23% (Fig. 6A6) in the liver and 5.38% (Fig. 6B6) in the kidney. The SEC + Cub-NPs–C. vulgaris (20) group revealed decreased liver ER-α levels averaging 10.43% (Fig. 6A7) and kidney levels averaging 2.62% (Fig. 6B7).

Notably, the SEC + Cub-NPs–C. vulgaris (40) group demonstrated a significant reduction in liver ER-α levels, averaging just 8.43% (Fig. 6A8), along with a markedly lower kidney average of 1.61% (Fig. 6B8), suggesting potent modulation of hormonal signaling pathways.

Ki-67 expression in the liver and kidney under varying treatments: The Normal Control group served as the baseline cellular activity with minimum Ki-67 levels (0.7% in liver (Fig. 7A1), 1.1% in kidney (Fig. 7B1)); whereas the SEC Treatment group showed higher Ki-67 levels of 21.4% in the liver (Fig. 7A2) and 27.8% in the kidney (Fig. 7B2) due to increased proliferation. TAM treatment resulted in 8.6% and 10.6% moderate reduction in Ki-67 expression from liver and kidney sections (Fig. 7A3 and B3, respectively).

The SEC+Polymer group treated group showed only marginal improvement (19.8% in the liver, 25.6% in the kidney), with Ki-67 levels remaining statistically comparable to the Tumor-Only group, indicating negligible therapeutic impact (Fig. 7A4 and B4, respectively). The SEC + C. vulgaris (20) and SEC + C. vulgaris (40) groups treatment brought down 12.9% in the liver and 16.2% in the kidneys (Fig. 7A5 and B5, respectively) and by 6.5% in the liver and by 8.1% in the kidney (Fig. 7A6 and B6, respectively), respectively.

Remarkably, the SEC + Cub-NPs–C. vulgaris (20) group showed limited expression at 5.2% in the liver and 6.1% in the kidney (Fig. 7A7 and B7, respectively), the least being for the SEC + Cub-NPs–C. vulgaris (40) group, 2.2% in the liver, and 2.3% in the kidney, showing the inhibition of proliferation in the higher concentration (Fig. 7A8 and B8, respectively).

The PCNA expressions in liver and kidney tissues provided insights into cellular proliferation across various treatment modalities. The Normal Control group demonstrated low PCNA expression, with mean values of 0.5% in hepatic tissue and 1.9% in renal tissue, establishing baseline levels of cellular proliferation (Fig. 8A1 and B1, respectively). In the opposite case, the highest increase was demonstrated in the SEC treatment group with series averaging 23.6% in hepatic and 30.0% in renal, with high proliferative activity due to tumor growth (Fig. 8A2 and B2, respectively). In contrast, the mean value was reduced to 10.2% in hepatic and to 17.6% in renal after TAM group treatment, presenting a mean 57% reduction compared to the untreated SEC group (Fig. 8A3 and B3, respectively).

The SEC+Polymer group showed only marginal improvement (22.7% in liver, 26.6% in kidney), with PCNA levels remaining statistically comparable to the Tumor-Only group, indicating a negligible therapeutic impact (Fig. 8A4 and B4, respectively).

The SEC + C. vulgaris (20) and SEC+Chara vulgaris (40) groups treatment yielded average PCNA expressions of 11% and 12% in the liver (Fig. 8A5 and B5, respectively), respectively, and 18.5% and 24.1% in the kidney (Fig. 8A6 and B6, respectively), reflecting a significant decrease in cell proliferation compared to the SEC group.

Lastly, the SEC + Cub-NPs–C. vulgaris (20) group and SEC + Cub-NPs–C. vulgaris (40) group achieved a decrease in PCNA expression levels, averaging 6.7% and 3.2% in the liver (Fig. 8A7 and A8, respectively), and 9.2% and 5.3% in the kidney (Fig. 8B7 and B8, respectively), suggesting that this nanoformulation proved to be much more effective in inhibiting tumor-associated proliferation.

VEGF expression in the liver and kidney under varying treatments: The Normal Control group exhibited minimal VEGF expression, with averages of 2.24% in the liver (Fig. 9A1) and 1.15% in the kidney (Fig. 9B1). The SEC group demonstrated markedly elevated VEGF levels, averaging 17.45% in the liver (Fig. 9A2) and 8.75% in the kidney (Fig. 9B2), indicating increased vascularization due to tumor presence.

TAM treatment resulted in 8.6% and 10.6% reduced VEGF expression, measuring 5.93% in the liver and 2.77% in the kidney sections (Fig. 9A3 and B3, respectively). The SEC+Polymer group exhibited only marginal improvement (15.82% in liver, 7.93% in kidney), with VEGF levels remaining statistically comparable to the SEC group, indicating negligible therapeutic impact (Fig. 9A4 and B4, respectively).

The SEC + C. vulgaris (20) and SEC + C. vulgaris (40) groups treatment yielded average VEGF expressions of 8.53% and 7.50% in the liver (Fig. 9A5 and B5, respectively), respectively, and 4.15% and 3.50% in the kidney (Fig. 9A6 and B6, respectively), indicating a moderate reduction in angiogenic activity compared to the SEC group.

Particularly, the SEC + Cub-NPs–C. vulgaris (20) group and SEC + Cub-NPs–C. vulgaris (40) group achieved the lowest VEGF expression levels, averaging 4.80% and 3.30% in the liver (Fig. 9A7 and A8, respectively), and 2.15% and 1.50% in the kidney (Fig. 9B7 and B8, respectively), suggesting effective inhibition of angiogenesis.

The caspase-3 expression analysis in the liver and kidney tissues of the experimental groups revealed significant variations; thus, the Normal Control group was the one that showed the lowest levels of this enzyme, with averages of 0.64% in the liver and 0.57% in the kidneys (Fig. 10A1 and Fig. 10B1, respectively). Conversely, the SEC group manifested an apparent increase of caspase-3 (8.29% in the liver and 9.66% in the kidneys), which implies that the process of apoptosis associated with tumor growth was more severe (Fig. 10A2 and Fig. 10B2, respectively).

TAM group medication led to elevated caspase 3 levels (16.00% in the liver and 13.76% in the kidneys) (Fig. 10A3 and Fig. 10B3, respectively), whereas the SEC+Polymer group showed caspase 3 levels comparable to the SEC group, with averages of 8.50% in the liver and 9.46% in the kidneys, indicating no significant improvement in apoptotic activity.

The SEC + C. vulgaris (20) and SEC + C. vulgaris (40) groups treatment yielded average caspase 3 expressions of 13% and 14.05% in the liver (Fig. 10A5 and B5, respectively), respectively, and 10% and 11.18% in the kidney (Fig. 10A6 and B6, respectively), indicating a moderate rise in apoptotic activity compared to the SEC group.

Both the SEC + Cub-NPs–C. vulgaris (20) group and SEC + Cub-NPs–C. vulgaris (40) group achieved the highest caspase 3 expression, averaging 23.94% and 30.10% in the liver (Fig. 10A7 and A8, respectively), and 21.76% and 46.96% in the kidney (Fig. 10B7 and B8, respectively), thus demonstrating a very effective apoptotic effect.

Immunohistochemical quantification differences in liver and kidney tissues among experimental groups were evaluated in Table 6 and 7.

Table 6.

Immunohistochemical quantification of liver tissues

Group	ER-α (%)	Ki-67 (%)	PCNA (%)	VEGF (%)	Caspase-3 (%)
Normal control	2.46 ± 0.55	0.70 ± 0.63	0.50 ± 0.82	2.24 ± 0.49	0.64 ± 0.12
SEC	35.04 ± 3.12	21.40 ± 3.45	23.60 ± 4.10	17.45 ± 2.85	8.29 ± 1.20
SEC + TAM	13.95 ± 2.10	8.60 ± 1.85	10.20 ± 2.00	5.93 ± 1.25	16.00 ± 2.50
SEC+Polymer	19.37 ± 2.85	10.70 ± 2.20	16.10 ± 2.75	10.03 ± 1.90	11.91 ± 1.80
SEC + C. vulgaris (20)	18.30 ± 2.40	12.90 ± 2.10	18.50 ± 2.60	8.53 ± 1.60	13.00 ± 2.00
SEC + C. vulgaris (40)	16.23 ± 2.10	6.50 ± 1.30	11.00 ± 2.00	7.50 ± 1.40	14.05 ± 2.10
SEC + Cub-NPs–C. vulgaris (20)	10.43 ± 1.80	5.20 ± 1.10	6.70 ± 1.50	4.80 ± 1.00	23.94 ± 3.20
SEC + Cub-NPs–C. vulgaris (40)	8.43 ± 1.50	2.20 ± 0.80	3.20 ± 1.00	3.30 ± 0.90	30.10 ± 4.00

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Liver markers were quantified using ImageJ (ER-α, VEGF, and Caspase-3) and field counts at high magnification (Ki-67and PCNA) across ten random high-power fields (mean ± SD, n = 10). Results show tumor tissues with ↑ ER-α, ↑ Ki-67, ↑ PCNA, ↑ VEGF, and moderate ↑ Caspase-3 compared to controls. Tamoxifen and free formulations achieved partial ↓ effects, while nanoformulations produced the strongest modulation (↓↓ ER-α, ↓↓ Ki-67, ↓↓ PCNA, ↓↓ VEGF, ↑↑ Caspase-3), reflecting reduced proliferation and angiogenesis with enhanced apoptosis

Table 7.

Immunohistochemical quantification of kidney tissues

Group	ER-α (%)	Ki-67 (%)	PCNA (%)	VEGF (%)	Caspase-3 (%)
Normal control	1.20 ± 0.40	1.10 ± 0.50	1.90 ± 0.70	1.15 ± 0.35	0.57 ± 0.15
SEC	17.87 ± 2.80	27.80 ± 3.90	30.00 ± 4.20	8.75 ± 1.80	9.66 ± 1.50
SEC + TAM	5.59 ± 1.20	10.60 ± 2.00	17.60 ± 2.80	2.77 ± 0.90	13.76 ± 2.20
SEC+Polymer	9.70 ± 1.80	13.00 ± 2.10	21.00 ± 3.00	5.15 ± 1.20	7.08 ± 1.00
SEC + C. vulgaris (20)	7.25 ± 1.50	16.20 ± 2.50	24.10 ± 3.20	4.15 ± 1.00	11.18 ± 1.80
SEC + C. vulgaris (40)	5.38 ± 1.10	8.10 ± 1.50	12.00 ± 2.00	3.50 ± 0.80	10.00 ± 1.50
SEC + Cub-NPs–C. vulgaris (20)	2.62 ± 0.80	6.10 ± 1.20	9.20 ± 1.80	2.15 ± 0.60	21.76 ± 3.00
SEC + Cub-NPs–C. vulgaris (40)	1.61 ± 0.50	2.30 ± 0.70	5.30 ± 1.20	1.50 ± 0.40	46.69 ± 5.20

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Kidney markers were analyzed using the same methodology (ImageJ for ER-α, VEGF, and Caspase-3; high magnification field counts for Ki-67 and PCNA; mean ± SD, n = 10). Tumor tissues showed ↑ ER-α, ↑ Ki-67, ↑ PCNA, ↑ VEGF, and moderate ↑ Caspase-3 relative to controls. Tamoxifen and free treatments yielded modest ↓ effects, whereas nanoformulations, especially at 40 mg, achieved maximal modulation (↓↓ ER-α, ↓↓ Ki-67, ↓↓ PCNA, ↓↓ VEGF, ↑↑ Caspase-3), indicating strong anti-proliferative, anti-angiogenic, and pro-apoptotic activity

Discussion

Bioactive substances, such as flavonoids, phenolics, saponins, tannins, alkaloids, and glycosides, are abundant in all algal groups. These compounds have significant antioxidant, anti-inflammatory, and antitumor properties [54]. C. vulgaris is less studied from the pharmaceutical and medical standpoints [12]. In the present study, numerous bioactive compounds were unraveled in the methanolic extract of C. vulgaris using GC-MS, indicating its possible potential pharmacological uses. Phytol (14.68%), oleic acid (13.92%), 1,2-benzene dicarboxylic acid (13.72%), hexadecanoic acid, 2,3-dihydroxypropyl ester (9.64%), hexadecanoic acid, methyl ester (4.67%), and 9-octadecenoic acid, 1,2,3-propanediol ester, (E, E, E)- (3.42%) were the most predominant components that have been found in the algal extract. These compounds have been connected to a number of biological processes [55, 56].

Cubosomes (Cub) are advanced lipid nanoparticles with a unique porous cubic structure that enables high drug loading and controlled drug release. The highly ordered bicontinuous cubic liquid crystalline nature of cubosmes gives them an advantages over liposomes [16]; cubosome is characterized by a significantly larger internal surface area, which confers substantially higher encapsulation efficiency for diverse drug molecules (hydrophilic, hydrophobic, amphiphilic) [16]; and enhanced physical stability that minimizes premature drug leakage and a tunable, sustained drug release profile that can be further modulated with surface coatings [17]. Cub offers superior gene delivery for cancer therapy compared to liposomes, effectively protecting nucleic acids like those used in RNAi and gene editing. Their structure allows for the co-encapsulation of genes and drugs within a single particle, enabling synergistic treatments that enhance overall therapeutic success [57].

Cub in the GIT maintains the encapsulated medication solubilized by entrapping it within the mixed micelles generated by cubosome digestion. Thus, they promote drug absorption, resulting in enhanced oral bioavailability [58]. It was reported that cubosomes may enhance intestinal absorption through two mechanisms. The first mechanism is through membrane fluidization, where their lipid components integrate into and disrupt the epithelial membrane, increasing paracellular and transcellular permeability. The second mechanism is via the promotion of trans-lymphatic transport. This second pathway facilitates direct entry into the systemic circulation, effectively bypassing first-pass hepatic metabolism and thereby increasing the bioavailability of the encapsulated therapeutic agents [59]. An earlier study produced a cubosome formulation, which successfully improved oral insulin absorption [60]. Another study revealed that Cub can boost the bioavailability of Coenzyme Q10, an antioxidant used to treat liver problems, by producing highly bioavailable and controlled drug formulations [61].

The ratio of Glyceryl Monooleate (GMO) to Poloxamer 407 was 5:1. The ratio was selected based on a synergistic combination of lipid phase structure and steric stabilization. Poloxamer 407 (a triblock copolymer of PEO-PPO-PEO) serves two essential functions: Steric Stabilization and inhibition of crystallization. Poloxamer 407 is adsorbed onto the surface of the GMO/water interface to prevent particle aggregation through the steric repulsion to enhance the stability of the cubosome. Additionally, it prevents GMO crystallization and keeps the formula in the desired crystalline state. Several studies reported the utilization of poloxamer 407 to act as a stabilizer in a concentration of up to 20% [16].

The preparation method was modified from previously published methods with slight modifications. It was found that increasing the GMO: poloxamer 407 ratio from 2:1 to 6:1 leads to the formulation of cubosomes with a smaller particle size, and on the other hand, increasing the ratio from 6:1 to 10:1 leads to a larger particle size. The author justified these results by the accumulation of poloxamer 407 at the ratio (2:1) at the bilayer of cubosome, leading to an increase in the steric effect resulting in larger particles, in addition to the liability of particles aggregation and vice versa, increasing the GMO: poloxamer from 6:1 to 10:1 leads to a decrease the stabilizing effect and steric effect of poloxamer [59].

UV spectrum of C. vulgaris showed a spectral peak ranging from 214 to 255 nm (ʎ_max 220 nm), 345–478nm (ʎ_max 410 nm), and 631–692 nm (ʎ_max 650 nm), indicating the presence of phenolic compounds [62–64]. Dynamic light scattering is a simple, non-invasive, rapid approach for the estimation of particle size in suspension. The main problem with DLS measurements is that heavier and bigger particles cause a polydisperse solution’s overall mean decay rate to be greatly influenced, which frequently results in an overestimation of these larger particles. The mean dynamic particle size ranged from 152.9 to 156.7 nm, meaning that the particle size was almost the same. These results were confirmed with the small value of PDI (0.26 ± 0.03). In liquid-crystalline systems, the zeta potential, which indicates the stability of colloidal dispersion, is another crucial characteristic that must be ascertained. It varied from − 24.32 to − 21.38 mV [65]. The SEM examination confirmed the monodispersion of the Cub-NPs–C. vulgaris; on the other hand, the TEM illustrated the actual hexagon shape (honeycomb) and size after utilizing uranyl acetate dye, with particle size ranging from 51.06 to 107.07 nm.

The FTIR spectrum of Cub-NPs–C. vulgaris showed a decrease in the percent transmittance for the peak at -3500/cm and the peaks at -2937 and − 2904/ cm, which could be elucidated by the formation of hydrogen; the specific peak of nitro compound at -1666/cm became sharper. This peak becomes sharper upon interaction with other compounds, indicating a change in the molecular environment or bonding characteristics. The sharpening of this peak often suggests increased order or crystallinity within the sample, which could be attributed to interactions with Poloxamer 407. Additionally, the peak of C–O stretching was shifted from − 1128 to − 1242/cm. This shift can be interpreted as a result of changes in hydrogen bonding or interactions with other components within a formulation. The sharper peak at − 1242/cm indicates a more defined and possibly stronger interaction involving the C–O groups, which may enhance the stability and functionality of Poloxamer 407 in various applications [66].

Incorporating patients with breast cancer with natural antioxidants is crucial because the development of cancer is linked to the generation of reactive species (ROS), which cause DNA damage, mutations, and chromosomal abnormalities that ultimately result in tissue disarray and injuries [67, 68]. Breast cancer development is also linked to the accumulation of ROS in women’s bodies [69], and the accumulation of ROS in the body alters the structure and functions of the liver and kidneys [70, 71].

The undifferentiated nature of solid Ehrlich carcinoma is comparable to that of human cancer [72]. Tumor development may endanger vital organs, particularly the liver and kidneys [4–6]. Here, our goal was to assess the effectiveness of C. vulgaris methanolic extract and its Cub nanoformulation in reducing the liver and kidney damage brought on by SEC as compared to standard tamoxifen chemotherapy.

GC-MS results showed the presence of various bioactive lipophilic compounds in C. vulgaris methanolic extract, namely phytol, oleic acid, 1,2-benzenedicarboxylic acid derivatives, hexadecanoic acid (palmitic acid) and its esters, and unsaturated fatty acid glycerides such as 9-octadecenoic acid (oleic acid) esters. Phytol, a diterpene alcohol, is a chlorophyll metabolite that has been extensively documented to exhibit potent antioxidant activity via free radical scavenging and redox regulation of cellular processes, aside from its established anticancer and pro-apoptotic activities. Unsaturated fatty acids, especially oleic acid and its glycerol esters, have been demonstrated to reduce oxidative stress by suppressing lipid peroxidation and regulating intracellular reactive oxygen species (ROS) concentrations, thus underpinning cytoprotective and antiproliferative activities. Additionally, other fatty acid esters such as hexadecanoic acid methyl and glycerol esters have been linked to antioxidant, anti-inflammatory, and membrane-stabilizing activities, which may have an indirect role in the suppression of oxidative stress and tumor development. Taken together, the co-occurrence of these compounds lends credence to the antioxidant activity of C. vulgaris methanolic extract and offers a rationale for its anticancer properties via the modulation of oxidative stress pathways [73–76].

In line with the conclusions of other earlier studies, our data demonstrated that SEC produced hepatic and renal dysfunction, as confirmed by raised serum ALT, AST, ALP, total bilirubin, urea, and creatinine [77, 78]. The histological analysis of hepatic and renal sections in untreated SEC-bearing animals revealed large nests of tumor cells demonstrating nuclear pleomorphism, abundant mitotic activity, vacuolar degeneration, and severe congestion, accompanied by an inflammatory cell presence, in the liver section and extensive tubular degeneration, glomerular atrophy, localized tubular necrosis, moderate inflammatory cell infiltration, and occasional tumor cells in the kidney section. These results parallel prior findings revealing the growth and spread of cancer into several internal organs [79, 80].

Mitochondrial damage [81, 82] or the detrimental consequences of tumor angiogenesis brought on by proangiogenic factor release may be the origin of the inflammatory cellular infiltrates that drive the advancement of hepatic and kidney inflammation [83].

This study establishes strong expressions of the ER-α, Ki67, VEGF, and PCNA, and weak expression of Caspase-3, found in the liver and kidney of SEC-bearing mice, by our immunohistochemistry analysis. These results were consistent with many other studies [81–83].

ER-α is expressed by 70–75% of breast cancer cells, which is associated with the degree of tumor estrogen reliance necessary for tumor growth and survival [80]. In the same context, PCNA and Ki67 are protein markers for cell growth and proliferation [84, 85]. Both healthy and malignant cells depend on PCNA proteins for DNA replication [86]. By creating a ring around it, it promotes and regulates DNA replication [87]. Also, G2/M exhibits the greatest levels of expression of Ki-67, a proliferating nuclear antigen that is produced in replicating cells during all cell cycle stages except for G0 [88].

In the same manner, the survival and relentless expansion of solid tumors like breast cancer depend on VEGF, a master regulator of angiogenesis [89]. Angiogenesis and apoptosis are out of balance in breast cancer cells [80]. Additionally, SEC-bearing mice were categorized by downregulated expression for key roles of apoptotic cascades, including caspase 3, P53, and BAX, which confirmed that cancer is characterized by downregulated apoptosis and enhanced cell development, which promote tumor growth and proliferation [89, 90].

Liver and kidney function indicators decreased in the groups treated with TAM, C. vulgaris (20 and 40 mg/kg), and Cub-NPs–C. vulgaris (20 and 40 mg/kg). Moreover, histological analysis of liver and kidney sections revealed comparable abnormalities, albeit less severe and dispersed. Furthermore, immunohistochemistry analysis for liver and kidney tissues showed a decline in ER-α, Ki67, VEGF, and PCNA expression and a rise in expression of caspase 3, which suggests a decrease in proliferation and a rise in apoptosis. Numerous investigations have revealed TAM’s anticancer action on breast cancer by apoptosis, angiogenesis inhibition, and estrogen receptor blockage [91, 92]. This study is the first one we are aware of to assess the potential effectiveness of cubosome nanoformulation of Chara vulgaris extract in reducing the liver and kidney damage brought on by SEC.

Conclusion

In summary, the findings suggest that treatment of SEC-bearing mice with C. vulgaris and Cub-NPs–C. vulgaris controlled serum levels of the changed parameters and improved the effects of ESC on hepatic and renal tissue structure, and ER-α, Ki67, VEGF, and PCNA protein expression, suggesting that C. vulgaris and Cub-NPs–C. vulgaris had a possible defensive effect against SEC-induced hepatic and renal tissue damage with special reference to Cub-NPs–C. vulgaris.

Acknowledgements

Acknowledgment: The authors extend their appreciation to Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R736), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Author contributions

Conceptualization, T.E.-M., and M.E.-N.; methodology, M.E.-N., M.G., and E.E.-Z.; validation, T.E.-M., E.E-Z, M.E.-S, M.G, A.S, E.E.-M, H.K.B, A.S.G, M.E.-N, M.A.-S, A. A.-R, and R.A.-Q; formal analysis, T.E.-M., E.E-Z, M.E.-S, M.G, A.S, E.E.-M, H.K.B, A.S.G, M.E.-N, M.A.-S, A. A.-R, and R.A.-Q; investigation, T.E.-M., E.E-Z, M.E.-S, M.G, A.A.S., E.E.-M, H.K.B, A.S.G, M.E.-N, M.A.-S, A. A.-R, and R.A.-Q; resources, T.E.-M., E.E-Z, M.E.-S, M.G, A.A.S., E.E.-M, H.K.B, A.S.G, M.E.-N, M.A.-S, A. A.-R, and R.A.-Q; data curation, M.E.-N., M.G., and E.E.Z.; writing—original draft, M.E.-N., M.G., E.E.-M and E.E.Z.; review and editing, T.E.-M., E.E-Z, M.E.-S, M.G, A.A.S., E.E.-M, H.K.B, A.S.G, M.E.-N, M.A.-S, A.A.-R, and R.A.-Q; funding acquisition, M.A.-S, A. A.-R, and R.A.-Q. All authors have read and agreed to the published version of the manuscript. The authors confirm that no paper mill or artificial intelligence was used.

Funding

Not applicable.

Data availability

The datasets generated during and/or analyzed during the current study are available within the manuscript.

Declarations

Ethics approval and consent to participate

The studies involving animal participants were reviewed and accepted by Tanta University’s, Faculty of Pharmacy, which approved laboratory animal handling practices that adhered to the updated Animals (Scientific Procedures) Act 1986 in the UK and Directive 2010/63/EU in Europe (Code of Protocol (TP/RE/4/23p-0019)).

Consent for publication

All authors have read and agreed to the published version of the manuscript.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available within the manuscript.

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PERMALINK

Evaluation on the manufacturing, properties, and potential use of Chara vulgaris extract cubosome nanoformulation to mitigate hepatic and renal damage from Solid Ehrlich carcinoma: in vitro and in vivo investigation

Maha Alsunbul

Thanaa A El-Masry

Enas I El Zahaby

Mostafa M El-Sheekh

Mohamed M S Gaballa

Abdullah A Saber

Eman E Elmohamady

Heba Kamel Mahmoud Badawy

Abeer Saad Gawish

Amal Abdullah Alrashidi

Reem ALQahtani

Maysa M F El-Nagar

Abstract

Graphical abstract

Introduction

Materials and methods

Drugs and chemicals

Collection of C. vulgaris biomass

In vitro study

Preparation of C. vulgaris extract

Methanolic extract of C. vulgaris analyzed using GC-MS

Preparation of nanoparticles

Characterization of Cub-NPs–C. vulgaris

UV spectrum of Cub-NPs–C. vulgaris

Drug content and % entrapment efficiency (%EE) of Cub-NPs–C. vulgaris

Particle size, zeta potential, polydispersity index (PDI)

SEM and TEM

FTIR investigation

In vivo study

Animals

Solid Ehrlich tumor induction

Design for experiment

Blood and tissue samples collection

Determination of liver enzyme

Kidney function assessment

Histopathological analysis

Immunohistochemical examination

Statistical analysis

Results

In vitro analysis

Examination of C. vulgaris methanolic extract by GC-MS

Fig. 1.

Table 1.

Characterization of Cub-NPs–C. vulgaris

Calibration curve and UV spectrum of Cub-NPs–C. vulgaris

Fig. 3.

Fig. 2.

Particle size, zeta potential, PDI, and drug content of Cub-NPs–C. vulgaris

Table 2.

SEM and TEM examination for Cub-NPs–C. vulgaris

FTIR analysis

Fig. 4.

In vivo analysis

Effect of Cub-NPs–C. vulgaris on liver and kidney function biomarkers

Table 3.

Table 4.

Histopathological analysis

Fig. 5.

Table 5.

Immunohistochemical examination

Fig. 6.

Fig. 7.

Fig. 8.

Fig. 9.

Fig. 10.

Table 6.

Table 7.

Discussion

Conclusion

Acknowledgements

Author contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes