A feasible strategy of fabricating camptothecin (SN38)-loaded holmium ferrite nanocarrier delivery for glioma treatment

Malignant gliomas are the most prevalent and deadly primary brain tumors. The life expectancy of people with gliomas only slightly increases through surgical procedures, radiation, and chemotherapy. Magnetic nanocarriers must be developed to enable drug delivery using a magnetic field. A utilized to fabricate holmium ferrite nanoparticles is described herein. β-Cyclodextrin-polyethylene glycol (PEG) conjugate is used as a coat for the holmium ferrite nanoparticles. X-ray diffraction, energy dispersive x-ray spectroscopy, and x-ray photoelectron spectroscopy are all used to study the nanoparticles. This size range of nanoparticles is optimal for efficient drug delivery. The in vitro cytotoxicity of the fabricated nanoparticles was examined using U87MG and LN229 glioma cancer cells. The acridine orange/ethidium bromide and nuclear staining methods examined the morphological changes in the U87MG and LN229 glioma cells. The mode of cell death mechanism was investigated by Annexin V-FITC/PI flow cytometry methods. The possibility for successful SN38 delivery for the treatment of glioma cancer exists with the SN38@HF-β-CD-PEG.


Introduction
Cancer is still the leading cause of death from the disease worldwide. Despite progress, the death rate from cancer remains disturbingly high. There were 3,30,000 new instances of central nervous system (CNS) malignancies in 2016, resulting in 2,27,000 fatalities [1][2][3]. Glioma is the most frequent primary central nervous system tumor, at a rate of 3-8 new cases per 1,00,000 persons per year (40%-50%). Gliomas are divided into four stages by the World Health Organization (WHO): Malignant brain tumors of grades I and II include astrocytomas, oligodendrogliomas, dysembryoplastic neuroepithelial tumors, gangliogliomas, and mixed gliomas [4]. Highgrade gliomas (malignant gliomas, MG) such as anaplastic astrocytoma, oligodendroglioma, ependymoma, gliosarcoma, and glioma are classified as WHO grades III and IV. More aggressive and with a worse prognosis is MG [5]. Less than 5% of patients diagnosed with grade IV survived for at least five years. Clinical treatment often consists of one or more standard methods: surgery, radiation therapy, or chemotherapy [6][7][8]. However, precise excision is challenging due to the tumor's infiltrative growth pattern [9]. Further, radiation and chemotherapy have significant and unfavorable effects on quality of life [10].
The FDA-approved anticancer drug irinotecan (camptothecin-11) is metabolized into the active metabolite SN38 (7-ethyl-10-hydroxyl camptothecin). Carboxylesterase-mediated cleavage in the liver converts inactive irinotecan to the bioavailable form SN38 after irinotecan intake [11]. So, in humans, only 1%-9% of an intravenous dosage of irinotecan gets changed into SN38. A hundred to a thousand times as effective as irinotecan is SN38. Its cytotoxic properties arise from its inability to perform topoisomerase I inhibition, an enzyme essential for properly regulating DNA replication and transcription [12]. Due to its high efficacy, SN38 is considered a treatment for numerous types of cancer, including lung, ovarian, breast, and colorectal. Since SN38 is not well soluble in water or other common pharmaceutical solvents, its therapeutic utility is limited [13]. Preventing the inactivation of anticancer medications of the camptothecin family by metabolism to the carboxylate form, which occurs under normal body conditions, is a significant challenge [14][15][16]. A tight lactone ring in SN38's structure makes it active at pH 5.0, but at physiological pH, the ring opens, transforming SN38 into an inert carboxylate system [17]. Therefore, therapeutic advantages and clinical efficacy must be achieved to design a formulation capable of retaining the active form of SN38, in addition to tackling the formulation difficulties of SN38 linked to its solubility in an aqueous solution [18].
Holmium orthoferrite (HoFeO 3 ), a member of the perovskite family with a distorted ABO3 orthorhombic structure of YFeO 3 type, is the focus of much recent research. HoFeO 3 's magnetic and dielectric behavior is due to the symmetry of its structure, which has led to its potential use in a wide range of industries and applications [19]. One such application is magnetotherapy products, which have effectively alleviated acute and chronic pain associated with several different injuries and fractures. In addition, HoFeO 3, as a dopant in different composite ceramics, has been the subject of much research [20]. It is well-known that the phase composition, structural characteristics, size, shape, and morphology of the resulting nanocrystals all play a significant role in determining the functional qualities of these materials [21]. Though there are many established techniques for making nanostructured ferrites and orthoferrites (including mechanochemical, sonochemical, hydrothermal, sol-gel, and other syntheses), achieving the desired functional properties of the resulting nanocrystals requires the use of sophisticated equipment and a wide range of synthesis parameters [22]. The powders of hexagonal orthoferrites of different compositions have been produced in various ways, as evidenced by numerous published publications on the subject [23]. In contrast to the strategies, the glycine-nitrate combustion process yields a powder in only a few minutes, has excellent phase homogeneity in the end product, and necessitates no elaborate or costly apparatus [24]. In contrast to other methods of producing rare-earth orthoferrites, solution combustion synthesis (SCS) has the added benefit of yielding both the orthorhombic modification and the metastable hexagonal form [25]. The authors achieved many hexagonal orthoferrite nanopowders, previously produced only by thin-film synthesis, using SCS [26][27][28]. Despite widespread interest in using glycine-nitrate combustion to make functional materials, no comprehensive studies have been conducted to determine how the combustion conditions affect the phase and chemical composition, morphology, structural, and magnetic features of hexagonal and orthorhombic HoFeO 3 [29][30][31].
Here, we describe the fabrication and characterization of holmium ferrite nanoparticles (NPs), their coating with a β-cyclodextrin (CD)-PEG conjugate, and the loading and release of the drug 7-ethyl-10-hydroxyl camptothecin (SN38), and an anticancer investigation of the SN38-loaded NPs against glioma cancer cell lines. The cytotoxicity and cellular internalization of the nanoformulation were studied in U87MG and LN229 human glioma cancer cells in vitro. The purpose of taking both U87MG and LN229 cells was to assess the efficiency of the proposed nanoformulation in glioma cells to gain a solid sense of the effectiveness of the nanoformulation for glioma cancer treatment. Various apoptosis staining methods examined the cancer cells' morphological properties. The final product's transmission electron microscopic (TEM) image was taken using a JEM-2100F transmission electron microscope (JEOL, Japan). Fourier transform infrared (FT-IR) KBr/sample pellet spectra were measured from a Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific, USA). The crystallization form was analyzed using an XRD-7000 x-ray diffractometer (Shimadzu, Japan) with CuKα radiation (λ = 1.5406 Å). Surface chemistry was determined by x-ray photoelectron spectroscopy (XPS) using an ESCALAB 250Xi x-ray photoelectron spectrometer (Thermo Fisher Scientific, USA). The nanoparticles' hydrodynamic size and zeta potential were measured through dynamic light scattering (DLS) via Zetasizer Nano ZS90 (Malvern Instruments, UK).

Experimental methods
The human glioma cancer cells (U87MG and LN229) were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). U87MG and LN229 glioma cancer cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin, 10,000 U ml −1 ) at 37°C in a humidified atmosphere containing 5% CO 2 .
2.2. Fabrication of holmium ferrite NPs (HFNPs) and β-CD-PEG conjugate A solution containing 1 M holmium nitrate and 2 M ferric nitrate was synthesized. The addition of ammonium hydroxide was followed by stirring with a magnetic stir bar. For the next 18-h, the solution was heated to 180°C within a hermetically sealed autoclave. The acetone was used to clean the solution and disperse the NPs that had been suspended in it.
PEG-bis-(tosylate) was synthesized according to a previously published method. Polyethylene glycol (1.0 ml), triethylamine (3.2 ml), and DCM (250 ml) were typically mixed at 45°C for 35 min We next added tosyl chloride (3.9 g) to 150 ml of DCM and stirred the reaction mixture for 4-h. Products were cleaned and dried. DCM was added to a mix of bis-sulfonate-PEG (1 g) dissolved in ammonia (30%) and agitated for 1-h. H 2 N-PEG-NH 2 was precipitated after the solvent was removed by evaporation and diethyl ether addition. In DMSO (5 ml), PEG-bisamine (3.0 g) and 6-p-toluenesulfonyl β-cyclodextrin (1.0 g) were heated to 65°C for 12h before being cooled and put into acetone. Filtration, acetone washing, and drying were all used to finalize the product. The HFNPs (10 mg) were added to 20 mg of the β-CD-PEG polymer in a slightly basic aqueous medium (pH 8.0, adjusted using NaOH).

Drug loading and release
On a microbalance, SN38-load HFNPs were weighed and dispersed in Phosphate Buffer Saline (PBS), maintained at pH 7.4. The concentration of SN38 was calculated by measuring its absorbance at 368 nm. The percentage of loaded drugs is given by %Adsorption efficiency Weight of SN 38 in HFNPs Weight of HFNPs 100 = -T he SN38-loaded NPs were suspended in dialysis bags, dipped in PBS solution (pH 7.4 and 6.0 separately), and a mechanical shaker was shaken at room temperature. The solutions released from the bags were withdrawn at pre-planned time intervals. The concentrations of the solutions were determined using UV-vis spectroscopy.
The average values obtained from three different intervals were utilized for plotting the drug release rate [32].
The in vitro SN38 release profile was studied, employing the previous method's reported method. SN38loaded HF-β-CD-PEG NPs (SN38@HFNPs) were put in dialysis bags with a molecular weight cut-off range of 1000 Da. The buffer solution PBS was utilized, and two different pHs, 7.4 and 6.0, were set up to measure the release of drugs of the pHs. UV-vis absorbance values were calculated for the solutions withdrawn.

Evaluation of in vitro cytotoxicity
The MTT analysis was performed to determine whether the optimized formulation (SN38@HF-β-CD-PEG) was cytotoxic to U87MG and LN229 cell lines and normal HEK293 l929 cell lines in vitro. Results were compared to those obtained with unconfined SN38. Cells were grown on a 96-well culture plate and DMEM culture medium (10 3 cells per well). After a 24-h incubation in an incubator, cell seeds were treated with free HFNPs, SN38, and SN38@HF-β-CD-PEG (at different concentrations) before being re-incubated for 24-h. Each cultured cell was removed after incubation. Following a 4-h incubation in MTT solution, cells were treated with dimethyl sulfoxide solution. The percentage of live cells was determined using a microplate reader at 540 nm. The viability percentage was examined by previous literature [33][34][35][36].

Morphological examination
The efficacy of SN38 and SN38@HFNPs to trigger apoptosis in U87MG and LN229 cells were assessed by the AO/EB dual staining approach. U87MG and LN229 cells were plated in 6-Well plates (10 6 cells) and incubated with HFNPs, free SN38, and SN38@HF-β-CD-PEG at IC 50 concentration. The plates were incubated for 24-h at 37°C with 5% CO 2 . After incubation withAO/EB dual staining, the cells were analyzed using a fluorescence microscope to determine apoptotic cell death [37][38][39].
Nuclear morphological alterations, such as fragmentation or condensation, linked with apoptosis can be evaluated using DAPI labeling. In this assay, U87MG and LN229 cells were seeded at a density of 10 6 cells well −1 in six-well plates before being treated with the indicated concentrations of HFNPs, free SN38, and SN38@HF-β-CD-PEG and incubated for 24-h at 37°C in 5% CO 2 . Following incubation, the cells were rinsed twice with PBS for 15 min. After incubation witha DAPI staining solution, the cells were analyzed using a fluorescence microscope to determine apoptotic cell death [40][41][42].
2.6. Apoptosis investigation by flow cytometry U87MG and LN229 cells were seeded at a density of 10 9 cells well −1 in six-well plates before being treated with the indicated concentrations of HFNPs, free SN38, and SN38@HF-β-CD-PEG and incubated for 24-h at 37°C in 5% CO 2 . After treatment with cells, 100 μl of staining buffer (5 μl of annexin-V and 5 μl propidium iodide (PI) were added to each flow cytometry tube and incubated at 4°C in the dark for 15 min. Samples were examined using the SYSMEX flow cytometer [43][44][45].

Results and discussion
3.1. Nanoparticles characterization Nanoparticles' pharmacokinetic profile and absorption in the blood are greatly improved when they are adequately sized and have their surfaces coated with organic or inorganic material. Particle size in the 10 to 200 nm range allowed for optimal penetration of cancer cells and increased cancer cell inhibition [46][47][48]. The potential therapeutic use of drug-loaded HFNPs (SN38@HF-β-CD-PEG) depends on their excellent drugloading efficiency, extended drug release, cytotoxic profile, cellular internalization, and morphological changes (figure 1).
β-CD-PEG was used to coat the NPs. X-ray powder diffraction was used to characterize the polymericcoated NPs (HF-β-CD-PEG) (figure 2(A)). It may be inferred from the sharpness of the peaks that the ferrite is created in a suitable crystallinity manner. The coated polymer produces a diffraction pattern with a curvature of less than 2ө 20°. No distinct holmium oxide phase is generated, as the peaks do not coincide with those previously described for Ho 2 O 3 . They are consistent with the XRD pattern for holmium-doped Fe 2 O 3 that has been described. In the presence of Ho 3+ , the lattice of  figure 2(C). The NPs' surface coating of thin polymer seems clear. The NPs appear somewhat spherical and measure 75±6 nm in size. The polydispersity index (PDI) and zeta potential of β-CD-PEG-HFNPs were displayed at 0.105±0.05 and −11±2.4 mV, respectively. As-prepared HFNPs EDX is shown in figure 2(B), and Ho, Fe, and O can all be displayed. No further lanthanide impurities were found in the HF. The ferrite is identified as HoFe 2 O 4 using ICP-AES. β-cyclodextrin-polyethylene glycol (β-CD-PEG) was used to coat the NPs. TGA results for the coated NPs are presented in figure 2(D). The HF-β-CD-PEG and SN38@HF-β-CD-PEG nanoparticles were stable under an aqueous solution and cell culture media on different days. The stability of the nanoparticles is an important issue for potential biological applications as well as for long-term storage and their possible use as candidate nanoscale reference materials. The stability of the fabricated nanoparticles was examined by the dynamic light scattering (DLS) measurement ( figure 3). This good stability is essential for using GNP probes in biological sample studies. Up to 180°C, the polymer releases water, resulting in a weight loss of around 10%. Up to 370°C, further weight loss of 18% occurs when the surface polymer melts. The subsequent   The FT-IR spectral analysis of the HFNPs is displayed in figure 5(A). The HFNPs appear to have a modest absorption band at 1350-1750 cm −1 . The β-CDPEG-coated HFNPs display absorption peaks from C-C stretching (1421 cm −1 ) and -C=N stretching (1631 cm −1 ). The Fe-O absorption peaks are found at 581 cm −1 . These show that the HFNPs are covered with a layer of β-CD-PEG. Additionally, the vibration and stretching bands at roughly 1650 and 3500 cm −1 are attributed to the stretching and bending vibration of the water peaks.

Drug loading and the release of drug
The anticancer drug SN38 was loaded in the carrier HF-β-CD-PEG (SN38@HFNPs) as per the procedure discussed in the experimental section. The drug loading percentage, determined by equation (1), is 92% UV-vis spectroscopy was employed to calculate the concentration of SN38, measuring the absorbance at 385 nm.
The in vitro release profiles of SN38 are shown in figure 5(C). The cumulative release of SN38 as a function of time reveals that the release is slow and sustained. The initial rapid release rate of SN38 becomes near constant after 20-h when the pH is 7.4. The cumulative release percentage is about 39%. A decrease in pH modulates the drug release. The cumulative release percentage increases, reaching a plateau of about 66% after 18-h. This result reveals that the release of SN38 is slow and sustained and occurs well above 48-h. Further, the release of the drug is tunable by varying the pH value.

Magnetic properties
The as-prepared HFNPs exhibit the sigmoidal magnetization curves ( figure 5(B)) characteristic of superparamagnetic NPs, with no hysteresis loop. We find a Ms value of 43 emu g −1 for the saturation magnetization. In other words, superparamagnetic NPs quickly relax when an external magnetic field is removed. It can be used for medication delivery aided by magnetic fields and magnetic hyperthermia therapy for cancer. Adding a β-CD-PEG coating on as-prepared HFNPs reduces the Ms and the magnetization shape [49][50][51].

In vitro cell viability assay
Assessing the NP's cytotoxicity toward cancer cells after production is essential for drug delivery. Figure 6 shows that the cytotoxicity of SN38@HF-β-CD-PEG, free HFNPs, and free SN38 was tested using an MTT assay against U87MG and LN229 glioma cancer cell lines. Cytotoxicity toward U87MG cells was lower for HFNPs without SN38 compared to SN38@HFNPs. More than 70% cell viability was displayed with HFNPs, but SN38@HF-β-CD-PEG demonstrated much higher toxicity than free SN38 or HFNPs at the same dose. For U87MG cells, the IC 50 value for SN38@HF-β-CD-PEG was determined to be 15.25±2.54 μg ml −1 . Comparatively, the toxicity of HFNPs to LN229 cells was much lower than that of U87MG cells. However, the cytotoxicity of SN38@HF-β-CD-PEG and SN38 on LN229 cells was much more significant. Similarly, U87MG and LN229 cells were more sensitive to SN38@HF-β-CD-PEG than free SN38 and HFNPs. For SN38@HF-β-CD-PEG, we measured an IC 50 of 16.29±2.87 μg ml −1 . The data from both cell lines imply that SN38@HFNPs may have clinical use in treating glioma cells. The cytotoxic effects of SN38@HF-β-CD-PEG, free HFNPs, and free SN38 on normal cell line HEK293 and L929 cells were also determined by the MTT assay. Cells were grown for 24 h and treated dose-dependent ( figure 7). The treatment of the highest SN38@HF-β-CD-PEG, free HFNPs, and free SN38 caused cytotoxicity on HEK293 cells, whereas treatment with high SN38 showed cell cytotoxicity observed. Overall, the fabricated nanoparticles don't affect the normal cells from this investigation.

Morphological examination of glioma cells
The AO-EB double-staining approach was used to examine the morphological alterations associated with apoptosis after NPs exposure. At 24-h at the IC 50 concentration, apoptotic characteristics such as condensed nuclei, membrane blebbing, and apoptotic bodies were seen in U87MG and LN229 cells treated with SN38, HFNPs, and SN38@HF-β-CD-PEG ( figure 8). Viable, early apoptotic, late apoptotic, and necrotic phenotypes were assigned to the labeled cells. Because of nuclear fragmentation and chromatin condensation, early apoptotic cells fluoresced a fluorescent solid green, while late apoptotic cells fluoresced an orange because of nuclear condensation, nuclear shrinkage, and blebbing [52]. The red necrotic cells were easily spotted. However, the nuclei of the control cells remained unchanged and resembled green (viable). The AO/EB staining outcomes reveal that the SN38@HF-β-CD-PEG efficiently kills the glioma cancer cells by observing fluorescence microscopy.
Cells were investigated independently using DAPI staining methods to establish that the apoptotic cell death produced by HFNPs, free SN38, and SN38@HF-β-CD-PEG was linked with nuclear condensation and fragmentation. U87MG and LN229 cells were stained with DAPI and evaluated under a fluorescence microscope following exposure to HFNPs, free SN38, and SN38@HF-β-CD-PEG for 24-h at the IC 50 concentration. Intercalating agent DAPI attaches to DNA in the spaces between individual bases. In this investigation, SN38@HF-β-CD-PEG produced nuclear alterations in cells undergoing apoptosis, as evidenced by the increased number of PI-positive cells ( figure 9). The results of DAPI staining demonstrate that the SN38@HFNPs shows efficiently kill the glioma cancer cells.  3.6. Apoptosis investigation by flow cytometry Cells treated with HFNPs, free SN38, and SN38@HF-β-CD-PEG were labeled with Annexin V-FITC and PI for flow cytometric analysis to determine the mechanism of cell death (figure 10). The transfer of phosphatidyl serine (PS) from the inner to the outside leaflet of the plasma membrane is a prominent feature of early apoptosis. Using fluorescently tagged Annexin V-FITC is a simple way to identify apoptotic cells because of Annexin V-FITC's strong affinity for PS. In contrast, PI is selectively permeable through damaged cell membranes, allowing it to detect necrotic cells. Figure 10 reveals that the percentage of early apoptotic cells in treated U87MG and LN229 cells with SN38@HF-β-CD-PEG reveals more significant apoptosis than in control cells. Late apoptotic and necrotic populations are not significantly different between treated and control cells in U87MG and LN229.

Conclusion
We present a holmium ferrite-mediated nanocarrier capable of delivery of 7-ethyl-10-hydroxyl camptothecin (SN38). The β-CD-PEG-coated HFNPs demonstrate a remarkable drug-loading capacity. The SN38 release from the nanocarrier is pH-dependent and prolonged. SN38 showed broad intracellular distribution on the U87MG and the LN229 cell lines, suggesting a biocompatible holmium ferrite nanocarrier composition was designed for prolonged and specific cell-targeted delivery of SN38 to glioma cancer cell lines. The formulation's efficacy in inhibiting the growth of human glioma cancer cells suggests it may be used in therapeutic settings. The acridine orange/ethidium bromide and nuclear staining methods examined the morphological changes in the glioma cells. These data support further investigation into the feasibility of SN38@HF-β-CD-PEG as a  delivery vehicle for SN38 in treating glioma cancer. However, more in vivo research on appropriate animal models is required before it can be successfully translated into clinical research.

Data availability statement
No new data were created or analysed in this study.

Author contributions
The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript.