Introduction

Heavy metal pollution limits the chemical synthesis techniques of magnetic nanoparticles (MNPs) due to their physiochemical properties and numerous applications such as drug delivery systems (DDS) or gene therapy systems (GTS)1. But; Green synthesis techniques has gained interest because of their nontoxicity, repeatability, scalability, and well-defined morphologies, which make them suitable for a wide range of applications2. Identifying new natural resources without negatively impact of food byproducts and waste like insects for the biological synthesis of MNPs is a key challenge in MNP synthesis3,4. Secondly challenge, there is a lack of knowledge regarding the chemical components and mechanisms for synthesizing MNPs5. The use of mealworms in our study effectively addressed both challenges; yellow mealworms (Tenebrio molitor L.) damage grain and flour economically6. But bioconversion of mealworms waste into high-value products is necessary for effective waste management and establishing a circular economy7. Therefore, it is essential to target the utilization of mealworms-based agri-food waste for the synthesis of value-added NPs products, especially Fe3O4, which may improve the role of DDS or GTS in cancer therapy8,9,10. Researchers use of these nanoparticles (NPs) for treatment of dangerous diseases such as cancer and optimization of drug or gene treatment to medical applications; for example, for targeting cancer cells with siRNA-FAM6,11. Some cancers can be treated with gene therapy. Currently, gene therapy uses antisense oligonucleotides and small interfering RNAs (siRNAs); DNA cannot easily cross cell membranes due to its size and anionic charge. With cationic polymers, DNA and RNA can be condensed into nanocomplexes12,13. Moreover, polycation coating protects siRNA-FAM against nuclease digestion. Polyethylene glycol (PEG) and Polyethyleneimine (PEI) are one of several polycations used to transfer genes to mammals14. Clinical applications of them are limited due to their toxicity15. Beside these, polyamines (PAs) regulate ion channels, nucleic acids, and protein synthesis, as well as regulating gene expression, protein function, macromolecule structure, and protecting the body from oxidative stress16. As a PA in the body, spermine is involved in the synthesis of proteins in mammalian cells; Furthermore, studies show that spermine levels are lower in people with disabilities17. Spermine also controls brain glutamate receptor activity, permits correct current flow through inward-rectifying K1 channels, protects against stress, and influences growth18. Spermine’s amine groups can neutralize DNA’s negative charge. Spermine is an appropriate delivery agent for oligonucleotides due to its good biocompatibility and ability to neutralize negative charges19. Due to spermine’s low molecular weight, it cannot optimally condense siRNA-FAM or DNA; hence, polyspermine (PS) synthesis is necessary to increase the molecular weight of spermine to deliver siRNA-FAM to mammalian cells19,20. According to one study, PS can bind genes and protect them from restriction enzymes20,21. The amine groups in PS and the phosphorus groups in siRNA-FAM interact electrostatically to form NPs11. PS shows great potential to protect DNA, and binds PEG- PS to stabilize NPs size for months19. Unlike PAs, polycations can also interact electrostatically with polyanions via amine groups through their positive charge. NPs are detected and removed by the Reticuloendothelial System (RES)22,23. Also, siRNA-FAM is released prematurely when it interacts with polyanions in plasma, since the polycation/gene complex is unstable24. NPs surfaces are coated with biocompatible, hydrophilic, and neutral polymers to increase their solubility14. Therapeutic potential is significantly enhanced by targeted gene and drug transfer25. In this way, side effects are reduced by minimizing gene and drug transfer. Targeted drug delivery to cancer cells involves using ligands that bind to receptors overexpressed on cancer cells’ surfaces26. Drugs are delivered more efficiently to target tissues when ligand-functionalized NPs are used27. During this study, we synthesized multitargeted NPs with a high ability to transfer genes into cancer cells and tissues while minimizing side effects on normal cells and tissues. Therefore, Fe3O4 NPs was extracted from the hydroalcoholic extract of food waste powder of the insect larvae mealworms (Tenebrio molitor L.) were exclusively fed on spinach leaves (Spinacia oleracea). To enhance spermine’s (SM) ability to encapsulate siRNA-FAM and DNA, hyperbranched spermines (HS) were synthesized; Then, Fe3O4- Hyperbranched Spermine (FHS) and Fe3O4-Spermine (FSM) were attached to PEG-folate in order to improve NPs half-life and target breast cancer cells with DNA and siRNA-FAM. Due to the high levels of folic acid on cancer cells’ surfaces, coating NPs with PEG-Folic acid enhanced siRNA-FAM or DNA delivery (Fig. 1).

Fig. 1
figure 1

Graphical abstract.

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Materials and methods

Materials

Materials used in this research were: 2-Carboxyethyl 2-Pyridyl Disulfide, FeCl3. 6H2O, FeCl2. 4H2O, (3-Mercaptopropyl) trimethoxysilane 95%, Tetraethyl orthosilicate, N-Hydroxysuccinimide 98% (NHS), Dimethyl sulfoxide (DMSO), Trioctylamine (TOA), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), Triphenyl phosphate, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT), Iron oxide (FeO) and Tetraethyl orthosilicate (TEOS), RPMI 1640, Fetal bovine serum (FBS), and NaOH; They were purchased from Sigma-Aldrich (USA). NH2-PEG-FA was purchased from nanocs (USA). Ammonia solution 25%, Aluminum chloride, Trichloroacetic acid, Thiobarbituric acid, Gallic acid, MgCL2, Tris-HCl, EDTA, Agarose gel, and Polyvinylpyrrolidone (PVP) were purchased from Merck (Germany). Pentane-1,3,5-tricarboxylic acid and Pyridine were purchased from Thermo Fisher Scientific (Canada).

Methods

Mealworm larvae

We used a constant climate chamber (HPP 110, Memmert, Schwabach, Germany), we raised yellow mealworm larvae aged eight weeks − 7th and 9th instar larvae - at 27 °C and 75% relative humidity; The larvae are provided by the Department of Plant Production of the University of Mohaghegh Ardabil, Iran. It is reared on two separate plastic culture media without any biological material that can be ingested by the larvae. Previously, our team studied iron-rich plants11,18; in one medium, spinach (Spinacia oleracea) was used as a food source to increase iron levels. To evaluate and compare iron levels another medium, wheat bran, was used as a common food source. As part of the study, spinach (Spinacia oleracea) leaves were fed to larvae until NPs were extracted. Mealworm larvae were separated from the spinach-food waste after eight-week period using a sieve (3.5 mm mesh). Spinach leaves were used to increase the iron content in Mealworm-based Spinach-Food Waste (MSFW), which includes insect feces6,28.

Preparing the hydroalcoholic extract

To prepare the hydroalcoholic extract, we added 10 g of MSFW powder (both MSFW powders, separately) to 100 ml of 80% methanol; Afterwards, we shaked the solution at 37 °C and 50 rpm for 24 h. Next, we centrifuged it for 10 min at 10,000 rpm. To remove the supernatant, filter paper (Whatman® 3MM Chr paper) was used18.

Synthesis of Fe3O4 NPs

A total of 3.33 g of iron (III) chloride hexahydrate (FeCl3. 6H2O) and 1.59 g of iron (II) chloride tetrahydrate (FeCl2. 4H2O) were placed into a 250-ml three-neck flask, followed by 100 ml of deionized water. At a suitable pressure, we blew nitrogen gas into the flask while attaching a thermometer to the neck. A 3 cm magnet was used to stir the contents of the flask after raising the temperature to 80 °C. A total of 15 ml of hydroalcoholic MSFW extract was added dropwise over a 10 min period (from each MSFW powder sample separately). After 5 min, 60 ml of NaOH (1 M) were added to the solution. We collected NPs using a magnet before washing them with deionized water to clean them. Table 1 shows the steps of synthesis. Our research used Fe3O4 extracted from larvae fed spinach (The results of the Table 1 were measured using an Atomic Absorption and with the help of a high temperature furnace)11.

Table 1 Mineral composition (mg/kg) of dried MSFW from both MSFW powder samples, separately; with schematic illustration of the synthesis of Fe3O4 NPs.
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Hyperbranched spermine (HS) synthesis

Pentane-1,3,5-tricarboxylic acid and amine groups in spermine can be combined to generate hyperbranched spermine. DMSO was used to dissolve pentane-1,3,5-tricarboxylic acid (1.5 mmol) and spermine (3 mmol) separately. To the reaction solution, 3 mL of pyridine was added together with 5 mmol of triphenyl phosphate, and the reaction was carried out for 12 h. In order to remove contaminants such as free spermine, a 3 day dialyse was performed against DMSO with deionized water (MWCO = 3500). A freeze dryer (Christ Alpha 1–4 LD plus, Germany) was used to dry the product (Fig. 2)29.

Fe3O4@SiO2-SS- SM and Fe3O4@SiO2-SS- HS NPs synthesis

According to Zhang et al., Fe3O4@SiO2-SS-SM NPs were synthesized in NH3.H2O, MPTMS, and TEOS solutions after sonication for 15 min in ethanol30. A magnet was used to separate the NPs, 5 rinses with ethanol and water were conducted (2 with ethanol and 3 with water), and the NPs were vacuum dried overnight. A sulfhydrylated Fe3O4 NPs solution of 200 mg in 12 mL Methanol and equal volumes of 2-Carboxyethyl 2-Pyridyl Disulfide was used for coupling Fe3O4@SiO2-SH with COOH. After 36 h, the product was dried and washed [10, 19]. To make Neither Fe3O4@SiO2-SS-SM nor Fe3O4@SiO2-SS-HS NPs, 200 mg of Fe3O4@SiO2-SS-COOH NPs were dissolved in 16 mL of PBS buffer (pH = 7.4). A combination of NHS (10 mg) and EDC (20 mg) was then used to activate Fe3O4@SiO2-SS-COOH carboxyl termini for 1 h. After mixing for 2 d at 25 oC, the combined solution was added to 4 mL of PBS buffer (pH ≈ 7.4) containing 200 mg of SM and HS. To dry the NPs in a freezer dryer, we rinsed them numerous times with deionized water and ethanol (Fig. 2)15.

Synthesis of Fe3O4-SM-PEG-FA and Fe3O4-HS-PEG-FA

In the synthesis of Fe3O4-SM-PEG-FA and Fe3O4-HS-PEG-FA, NHS-PEG-FA was bound to Fe3O4-SM and Fe3O4-HS. The mixture was stirred at 25 °C for 2 h after 100 µg (500 µg and 1000 µg) of Fe3O4-SM NPs and Fe3O4-HS NPs with NHS-PEG-FA 25 µg (125 µg and 250 µg) NPs were added. A centrifuge (15000 rpm for 15 min) was used to collect Fe3O4-SM-PEG-FA and Fe3O4-HS-PEG-FA, followed by washing several times with deionized water and drying under vacuum.

Fig. 2
figure 2

Schematic illustration of the synthesis of: Iron-Spermine (FSM), Iron-Hyperbranched Spermine (FHS), Iron-Spermine-Polyethylene Glycol-Folic acid (FSMPF), and Iron-Hyperbranched Spermine-Polyethylene Glycol-Folic acid (FHSPF).

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Preparation of the FSM/DNA, FHS/DNA, FSMPF/DNA, and FHSPF/DNA

Using the double emulsion solvent evaporation technique, Fe3O4-SM (FSM), Fe3O4- HS (FHS), Fe3O4-SM-PEG-FA (FSMPF), and Fe3O4-HS-PEG-FA (FHSPF) NPs were added separately to a fixed amount of DNA (5 µg) and siRNA-FAM (5 µg) using different concentrations of NPs (100, 500, 1000) (Table 2)32. Briefly, 5 µg of DNA (pEGFP-N1)15 or siRNA (siRNA-FAM) (0.1 ml TE) was added to variety of concentrations of copolymers (4 ml of 50% acetone + 50% DCM) (Table 2). A sonicator® XL (Misonix, NY) was used to sonicate the mixture for 30 s at 0 °C. We then added 6 ml of PVA solution (1% w/v) to the emulsion and sonicated it for 2 min at 0 °C to emulsify it. In 30 ml of PVA solution (0.3% w/v), the mixture was poured dropwise under mechanical stirring. In order to collect the NPs, we centrifuged them at 16,602 ×g for 30 min while removing the solvents (acetone and DCM) using a rotary vacuum evaporator (Heidolph). We then lyophilized the NPs by freezing them. In addition, 0.1 and 0.45 m membrane filters (SKC MCE Membrane Filters) were used. Assisted by this, FSM, FHS, FSMPF, and FHSPF were removed from aggregates. NanoDrop (Thermo Scientific 2000, USA) was used to measure the amount of DNA in the supernatant of each sample at 260 and 280 nm and compare it to the amount of DNA used for NP preparation (Eq. 1). Based on data obtained from this team’s previous results on SM and Fe-SM; DLS analysis revealed Fe3O4 nanoparticle sizes of 17 ± 3 nm, which increased to 23 ± 16 nm after spermine coating. Concurrently, the surface charge significantly increased from − 3.2 ± 0.35 mV to 18.42 ± 3.2 mV upon coating (Additional information in the Supplementary Information file - Figs. 1 and 2SI).

$$\left( {\% {\text{DEE}}} \right) = \frac{{{\text{~Amount~of~DNA~in~NP~}}}}{{{\text{Amount~of~DNA~used~for~NP~preparation}}}} \times 100$$
(1)
Table 2 Chemical composition of FSM/DNA, FHS/DNA, FSMPF/DNA, and FHSPF/DNA NPs; with schematic illustration of preparation.
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Characterization of NPs

Hydrogen nuclear magnetic resonance spectroscopy (1 H-NMR-Bruker 400 MHz) and Fourier transform infrared spectroscopy (FTIR-ABB Bomem-MB) were used to characterize NPs. Transmission electron microscopy (TEM, JEM-2100) and dynamic light scattering (DLS, Malvern Instruments, Westborough, MA, USA) were used to determine the morphology, particle size, and zeta potential of the NPs. A vibrating sample magnetometer (VSM, Standard 7403 Series, Lakeshore, OH, USA) was used to examine the magnetic properties of Fe3O4, Fe3O4-SM, Fe3O4-HS, Fe3O4-SM-PEG-FA, and Fe3O4-HS-PEG-FA NPs. In order to determine the thermogravimetric properties of NPs (TG-DTA-32, Japan), a heating rate of 25 °C to 600 °C was used, and a device EDX-7200 was used to measure the elemental content of the NPs (Energy Dispersive X-ray Fluorescence Spectrometer).

Retardation assay

Agar gel electrophoresis was used to test the ability of our NPs to neutralize DNA’s negative charge. Additionally, agarose gel electrophoresis was used to identify the appropriate ratio of NPs to DNA for in vitro and in vivo gene transfection experiments. The resulting complexes (20 µl of each of the resulting complex) were electrophoresed for 1 h at 80 V on 0.8% agarose gel. Our MNPs/DNA were compared with PEI/DNA (Additional information in the Supplementary Information file - Fig. 3_SI).

DNA protection assay

After the 0.8% agarose gel electrophoresis, NPs were tested for their ability to protect DNA against DNase I enzyme. As described above, NPs were first prepared for this purpose. A DNase I enzyme (2 U/µg of DNA) enzyme was then added to the NPs along with a 10 mM MgCl2 solution and a 50mM Tris-HCL solution at 37 °C. After 1 h of incubation, EDTA solution (3 µl, 1 M, pH 8.0) was added to inactivate enzyme activity. Then, MNPs were separated from DNA using Navarro et al.‘s method33. Our MNPs/DNA were compared with PEI/DNA and control DNA (0.8% agarose gel, 80 V for 1 h) using agarose gel electrophoresis.

In vitro cytotoxicity assay

Using MTT assay of PEI (control), FSM3, FHS2, FSMPF1 and FHSPF2 NPs were tested for cytotoxicity based on their similarity in size, surface charge, and DNA negative charge neutralization. 96-well plates filled (cell density of 7 × 103 cells per well) with MCF-7 cells were incubated at 37 °C with 5% CO2 in 200 µL of complete medium (RPMI 1640 medium containing 10% FBS). A total of 12 h, 24 h, 48 h, and 72 h were spent incubating cells at 37 °C with different concentrations of PEI (control), FSM, FHS, FSMPF, and FHSPF NPs. A MTT assay was then performed at 570 nm (BioTek Instruments; Winooski, VT, USA) to determine cell viability34. Finally, based on the following equation (Eq. 2), the percentage of viable cells was calculated:

$${\text{Cell viability }}\left( \% \right) = \frac{{{\text{OD~of~sample~at~}}\;570{\text{~nm~}}}}{{{\text{OD~of~control~at~}}\;570{\text{~nm}}}} \times 100$$
(2)

In vitro DNA delivery

Due to their similarity in size, surface charge, and neutralization of DNA negative charge, FSM3/DNA, FHS2/DNA, FSMPF1/DNA, and FHSPF2/DNA NPs were prepared in the same way as described above. FSM3, FHS2, FSMPF1 and FHSPF2 NPs were evaluated for their ability to deliver DNA to cancer cells using MCF-7 cells. 1 ml of RPMI 1640 culture media containing 10% FBS and 1% Penicillin/Streptomycin was added to each well of the 24-well plates containing MCF-7 cells (1 ml, 2 × 104 cells per well). Incubation of the plates was performed at 37 °C, 5% CO2 and 95% relative humidity for 24 h. NPs were tested in two different media with or without 10% FBS to determine whether they could deliver DNA to MCF-7 cells. To achieve this, the cells were washed with phosphate buffer (PBS, pH 7.3) and treated with NPs containing DNA (5 µg) in two media containing or not FBS (10%). As described above, the cells were incubated for 7 h. As positive and negative controls, PEI/DNA (N/P = 5, 5 µg DNA) and DNA (5 µg) were used. A fluorescence microscope (Nikon TE200) was used to examine the cells after three washes of phosphate buffer (PBS, pH = 7.4). Data collected using FloMax software (Version 2.3, 03.05.2001) was also analyzed quantitatively using flow cytometry (CyFlow Space, Germany). Luminometer (Brthlod Detection System, Pforzheim, Germany) was used to measure Luciferase expression (luciferase was quantitatively determined using Promega Luciferase Assay Kit) in MCF-7 cells lysed with 50 µL of cell lysis buffer.

Release kinetics and NP stability in presence of dextran sulfate

FSM3/DNA, FHS2/DNA, FSMPF1/DNA, and FHSPF2/DNA were selected due to their similarity in particle size and zeta potential to PEI/DNA complex.Various DNA complexes were prepared as described above for dextran sulfate assays, including PEI/DNA complex (N/P = 5; PEI: DNA mass ratio of 0.5:1), FSM3/DNA, FHS2/DNA, FSMPF1/DNA and FHSPF2/DNA. In addition, experiments were performed with dextran sulfate concentrations of 10, 20, 30, 40, and 50 mg/ml in different media containing different pH and ionic strengths to compare stability. For determination of release kinetics; It was dissolved in a sodium acetate buffer (pH 4.5) and a 5% glucose solution (pH 7.4). Sodium acetate buffer (pH 4.5) and a 5% glucose solution (pH 7.4) were used to boil separately the PEI/DNA complex, FSM3/DNA, FHS2/DNA, FSMPF1/DNA and FHSPF2/DNA at 37 °C in order to determine the release kinetics. The NPs were collected at scheduled intervals (30 min, 1 h, 2 h, and 3 h), centrifuged (16602 ×g, 30 min), and the supernatants were used for the release assay. Incubation at 37 °C was then carried out until the next interval by resuspending the NPs in 20 mL of fresh sodium acetate buffer or 5% glucose solution (pH 7.4). A NanoDrop spectrophotometer was then used to determine the DNA concentration in each sample (Eq. 3) 35.

$${\text{Cumulative release percentage}}\left( \% \right) = \frac{{{\text{Total~DNA~content~in~supernatant~of~each~samples}}}}{{{\text{The~amount~of~DNA~encapsulated~into~NPs}}}} \times {\text{100}}$$
(3)

In vivo transfection assay

The animal study was performed in two time periods (2 d and 30 d) and in separate groups. A transfection assay was conducted using female BALB/c mice in the present study. University of Mohaghegh Ardabili’s Ethics Committee approved this study and guided its conduct. We prepared BALB/c mice (body weight, 25–30 g) carrying breast cancer according to our previous report36,37. Using siRNA-FAM Control, PEI/siRNA-FAM, FSMPF1/siRNA-FAM, FHSPF2/siRNA-FAM, and PBS (the amount of RNA was 50 µg/kg for each mouse), each mouse’s tail vein was then injected. Tumors were harvested from the mice 48 h after injection. The siRNA-FAM concentration of tumor samples was determined based on the previous report38 after three rounds of phosphate buffer washing. Furthermore, FSMPF1 and FHSPF2 NPs were used to investigate the effect of an external magnetic field on siRNA-FAM delivery efficiency in tumor tissue using a neodymium magnet. The Lung, Spleen, Liver, Heart, Kidney and Tumor were extracted and homogenized in PBS. Luciferase activity in all of this were measured using FB 12 Luminometer (GmbH, Germany). Transfection efficiency of Tissues for PEI/DNA, FSMPF1/DNA, and FHSP2/DNA MNPs were expressed as relative light units RLU/mg tumor. We used carbon dioxide (CO2) for euthanasia as a chemical method.

In vivo toxicity analysis

In healthy female BALB/c mice (body weight, 25–30 g), the toxicity of NPs was evaluated by monitoring body weight and survival rates. In each group, mice were randomly divided into four groups (with 5 mice in each): PBS group, PEI/DNA, FSMPF1/DNA group, FHSPF2/DNA group, and mice without treatment as control. Injection of 100 µl of the samples (50 µg of DNA per mouse) was performed intravenously into the tail vein of the mice every 7 h for 3 d. Daily weight loss of the mice was recorded for 30 d (We used carbon dioxide (CO2) for euthanasia as a chemical method). According to the following equation, body weight was determined as a percentage (Eq. 4):

$${\text{The percentage of}}\;{\text{body weight}} = \frac{{{\text{W}}0 - \left( {{\text{W}}0 - \left[ {\frac{{{\text{W}}1}}{{\text{N}}}} \right]} \right)}}{{{\text{W}}0}} \times 100$$
(4)

W0, Represent the initial average body weight of the mice in each group before treatment with the NPs; W1, Represent the total average body weight of the mice in each group at different time points after treatment with the NPs; N, The number of measurement time points after treatment with the NPs.

Statistical analysis

The quantitative traits were replicated three times. A 5% probability level Duncan’s multi-range test was used to compare means. SPSS 16 and Excel were used to carry out the statistical analysis. Data acquisition was performed using FloMax software, version 2.3, from March 5, 2001. We then calculated the standard deviation of each treatment’s means. In order to conduct the survival test, Kaplan-Meier method was used to calculate the survival rate for mice in each group.

Results and discussion

Preparation and characterization of the NPs

NMR analysis of copolymers (SM, HS, PEG- Folic acid) and Characterization of Fe3O4 (NPs) extracted from MSFW

In a first step, the structural components of spermine, hyperbranched spermine (HS), and NHS-PEG-FA were elucidated using1H NMR analysis. As shown in Fig. 3, image A1, H NMR spectra of spermine showed peaks at 1.7 and 2.6 ppm related to hydrogen (a) and spermine (b). We observed some new weak peaks around 7.7 and 6.7 ppm after synthesizing hyperbranched spermine. According to Fig. 3, image B, these peaks result from pentane-1,3,5-tricarboxylic acid containing hydroxyl groups and ONH in HS containing hydrogen bonds11. Also, the peaks at 6.1 ppm and 4.2 ppm can be attributed to hydrogen in the hydrogen bond between NHS-PEG-FA and FFD39. Additionally, the small peaks at 6.7–8.5 ppm are attributed to the aromatic protons of folic acid (Fig. 3, image C)18. A series of chemical tests continued with XRD, magnetic behavior, transmission electron microscopy (TEM), scanning electron microscopy (SEM), TGA, and FTIR spectrophotometry in order to confirm the chemical structures of the Fe3O4 (NPs) extracted from MSFW. Based on Fig. 3, the peaks at 575 cm-1 correspond to the tensile vibration of the octahedral Fe-O structure, while the peak at 2931 cm-1 corresponds to the tensile vibration of the C-H bond. It is confirmed that Fe3O4 compounds exist from the peak at 575 cm-1. Aside from this combination of iron, no other iron combination has been synthesized, such as goethite or hematite. According to Fig. 3, the material contains fingerprint regions of glucopyranoxylamine or glucopyranose rings within the 1000–1155 cm − 1 region related to C − O and C − O−C bonds. A couple of bands of amide I and II in chitin (1555, 1620, and 1660 cm − 1) were found as well40,41. The peaks at 1630 and 1530 cm − 1, associated with amide bonds in proteins11,18, predominated in Fe3O4 (NPs) obtained from MSFW samples (Fig. 3). Additionally, Miller indices of XRD peak values were compared, i.e., 220, 311, 400, 511, and 440, which respectively correspond to the angles 30.9º, 35.9º, 43.8º, 57.7º, and 62.9o observed in Fe3O4; Profiles showed the typical pattern of α-chitin, with a peak of maximum intensity at 19.2° and a second peak at 9.2°, which is in accordance with results reported for insect chitin. There is a peak at 22.5° corresponding to the cellulose Iα plane. It is clear from the XRD peaks that the synthetic NPs are small39. The crystalline structure of Fe3O4 NPs was examined using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and XRD techniques. Fe3O4 NPs (Fig. 3, images H, F, and I) have a spherical shape, a smooth surface, and a non-uniform distribution. Human body dispersibility of NPs is influenced significantly by their shape. In addition to their properties, NPs morphology has a significant impact on them. The transfer rate of spherical NPs to cells is higher than that of other NPs morphologies, according to past research. According to Image J measurements, the NPs measured between 10 and 20 nanometers in size. According to Fig. 3, image A, Fe3O4 NPs have a saturation magnetization of 46.8 emu/g. As reported previously, these data are consistent11,39,40. In our final analysis, thermometric analysis confirmed the synthesis of Fe3O4 NPs extracted from MSFW. Figure 3 shows the TGA curves for Fe3O4. Fe NPs degraded in two steps based on TGA analysis. It is possible that Fe3O4 loses mass at temperatures between 50 and 100 °C as a result of water molecules that are adsorption on its surface. Furthermore, Fe3O4 NPs degrade between 100 and 300 °C in the second stage of degradation. It is possible that some of the unstable compounds in Fe3O4 NPs are responsible for this15,42.

Fig. 3
figure 3

1H-NMR spectroscopy of (A) Spermine, (B) hyperbranched Spermine, and (C) NHS-PEG-FA; FTIR characterization (D), TGA characterization (E), TEM characterization (F), Magnetic behavior characterization (G), XRD characterization (H), and SEM characterization (I) of Fe3O4 (NPs) extracted from MSFW.

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Characterization of FSM, FHS, FSMPF, and FHSPF copolymers

According to Fig. 4, Fe3O4@SiO2-SH, FSM, FHS, FSMPF, and FHSPF show their FTIR spectra. The peak at 11,050 cm-1 indicates that the Si-O bond of Fe3O4@SiO2-SH NPs is responsible for the Fe3O4@SiO2-SH peak. There is a FTIR spectrum presented of spermine and HS. Spermine’s peaks between 3200 and 3500 cm-1 indicate the presence of N-H (primary and secondary amines) vibrations. At WN 3200–3500 cm-1 and 1690 cm-1, representative bands were seen in the HS spectrum, associated with C-N and C = O groups of the HS (Fig. 4, images D). Fe3O4@SiO2-SS-SM and Fe3O4@SiO2-SS-HS FTIR spectra showed peaks at WN 1645–1755 cm-1, confirming SM and HS conjugate with Fe3O4@SiO2-SS-COOH. When Fe3O4@SiO2-SS-COOH are conjugated with SM and HS, peaks from WN 1645 cm-1 to 1755 cm-1, which correspond to the amino groups of the amide and HS, indicate that the SM and HS are attached to Fe3O4@SiO2-SS-COOH NPs (Fig. 4, images E and F). PEG-folate bound to HS were also confirmed using FTIR spectroscopy. It appears that PEG-related peaks are present in both HSPF and HSPG, suggesting they have been grafted with PEG. A peak of WN 1645 cm-1 to 1755 cm-1, attributed to the amide and amide amino groups, indicates attachment of the FSM and FHS to the surface of PEG-FA NPs after conjugation with PEG-folic acid (Fig. 4)11,15,18,39,40. Peaks related to PEG were observed at approximately 948 cm − 1 (C-H) and 1348 cm − 1 (C-O-C) for the Fe3O4@SiO2-SS-SM NPs, indicating that PEG was successfully grafted to the NPs43. PEG covalent binding has one weakness: it interferes with DNA release inside the cell, thereby increasing the possibility of the DNA being destroyed in the endosome44. Due to their high solubility in aqueous media, hydrophilic molecules like DNA encapsulate poorly within the hydrophobic nucleus45,46. In addition to PEG, Hyperbranched Spermine, Spermine, and Folic acid, there are other techniques for encapsulating DNA into hydrophobic polymers. The solvent evaporation technique was used in this study to encapsulate DNA. It encapsulates smaller particles and is more efficient than other techniques. The size and intrinsic potential of particles also play an important role in drug and gene delivery, with some cells only able to transfer particles below a micrometer in size47. Particles in the circulatory system live longer if their size is reduced and their gene transfer efficiency is improved, reducing the possibility of them being detected by the reticuloendothelial system. In addition to NPs morphology, experimental and computational studies have been conducted on gene transfer18. MNPs with spherical structures have been shown by previous research to be highly transferable to cells. As well as being more difficult to detect by the immune system, spherical MNPs are more stable in the circulatory system than other forms of MNPs15. A spherical structure was observed in the MNPs of FSM3/DNA, FHS2/DNA, FSMPF1/DNA, and FHSPF2/DNA as observed by transmission electron microscopy (Fig. 4).

Fig. 4
figure 4

FTIR spectra of Fe3O4 (A), Fe3O4-SiO (B), Spermine (C), Hyperbranched spermine (D), FSM (E), FHS (F), PEG-FA (G), FSMPF (H), and FHSPF (I) NPs.

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Figure 5 shows the magnetic behavior of FSM, FHS, FSMPF, and FHSPF copolymers, as well as their TGA, EDX, and UV-Vis measurements. For Fe3O4 NPs, the saturation magnetization was 41.3 emu/g (Fig. 5, image A). The data are in agreement with those in a previous report. After being coated with FSM, FHS, FSMPF, and FHSPF, Fe3O4 NPs saturation magnetization decreased significantly from 46.8 emu/g to 40.2 emu/g, 40.2 emu/g to 38.1 emu/g, 38.1 emu/g to 25.8 emu/g, and 25.1 emu/g to 20.9 emu/g. When MNPs are encapsulated in biodegradable NPs (encapsulation of MNPs into biodegradable NPs), saturation magnetization is often reduced11,18,36.

As shown in Fig. 5, image B, TGA curves for Fe3O4, SM, HS, FSM, FHS, FSMPF, and FHSPF have been obtained. TGA analysis revealed two steps of degradation for Fe3O4 NPs, but several steps of degradation were observed for FSM, FHS, FSMPF, and FHSPF NPs. Therefore, it may be concluded that structures such as FSM, FHS, FSMPF, and FHSPF contain more compounds than Fe3O4 NPs. For both Fe3O4, Fe3O4-Si, FSM, FHS, FSMPF, and FHSPF NPs in the range of 50 to 100 °C and for both SM and HS NPs in the range of 50 to 200 °C and 400 to 300 °C, the trend in weight loss is similar as shown in Fig. 5, image B. Consequently, after Fe3O4 was modified with SM, HS, and PEG-FA, its temperature resistance decreased significantly. As can be seen in Fig. 5, image B, Fe3O4, SM, PEG-FA, and HS have successfully conjugated6,11,18.

Figure 5. Image C shows a UV/ Vis curve for Fe3O4 NPs synthesized with spinach extract. Fe3O4 is synthesized and stable, as demonstrated in image C. A remedy for weakening the power of rays passing through a solution is to increase absorption and reduce crossing. Additionally, Fe3O4 can be synthesized with nanometric dimensions due to the absorption peak. SM, HS, and PEG-FA did not show any visible peak in the UV/VIS results, which was consistent with previous studies. The UV/Vis spectra showed that Fe3O4-Si peaks at 360–400 nm were sharply reduced; FSM, FHS, FSMPF, and FHSPF peaks at 330–500 nm sharply decreased because of the binding of spermine on the Fe3O4@SiO NPs surface (Fig. 5, image C). In FSM, FHS, FSMPF, and FHSPF, the slope indicates high sensitivity (the slope of the line of highest fit indicates sensitivity); therefore, the slopes of the lines indicate high sensitivity. The energies N, C, Fe, and O were shown to be present in the structure of synthesized NPs by energy dispersive X-ray spectroscopy (EDX) analysis (elemental analysis). Furthermore, the structure of synthesized NPs for both spermine (SM) and hyperbranched spermine (HS) contains elements N, C, and O. Related to Fig. 5, images D, E, F, G, H, I, J, and K, these results are consistent with those of previous reports11,15,18,40,41,43. A nanocarrier’s biocompatibility is an important criterion for transferring drugs and genes into damaged tissues. Nanocarriers with high toxicity have limited their use in clinical applications despite their potential for drug delivery to eukaryotic cells48. DNA is easily condensed and transferred to eukaryotic cells by our NPs. Therefore, different combinations of polymers have been used to prepare nano vectors to achieve a combination of advantages as well as to cover each other’s disadvantages. In this study, PEG, Spermine, and Folic acid were found to significantly reduce polymer toxicity by coating the surface with them19. A number of studies have demonstrated the ability of cationic nanopolymers to transfer genes to eukaryotic cells45. By neutralizing and compressing DNA’s negative charge, our polymer enhances gene transfer efficiency. Molecular weight, high surface charge, and polymer chain flexibility are all factors involved in cationic polymer compression of DNA43,49. It also creates the effect of a proton sponge, which protects DNA from endosome conditions. Preparing DNA-PEI complexes with N/P ratios has a significant impact on the efficiency of gene transfer. It won’t be able to transfer genes at low N/P ratios, but it will also result in a large and unstable complex15. An amine group in spermine is highly capable of interacting with the phosphorus groups in DNA and neutralizing their negative charge. Since spermine has a low molecular weight, it cannot optimally condense DNA. Therefore, PS synthesis is necessary in order to increase the molecular weight of spermine in order to enhance DNA delivery to mammalian cells. The greater the N/P ratio, the greater the toxicity, and the greater the barrier to gene transfer created by excess PEI15,50. In PAs, an amine group is positively charged, but in polycations, an amine group may interact with a polyanion in the plasma through an electrostatic relationship. In response, the NPs increase in size, causing the reticuloendothelial system to detect and remove them. Additionally, DNA will be released prematurely before it reaches the target tissue when the polycation/DNA complex interacts with the polyanions in plasma51. Biocompatible, hydrophilic, and neutral polymers (such as PEG, Folic acid, etc.) can be used to functionalize NPs surfaces and increase their solubility within the circulatory system52,53. In order to develop novel and effective therapeutic techniques, surface functionalization of NPs might be an interesting strategy to enhance current clinical disease diagnosis. Drug delivery efficiency and side effects are improved by using folic acid as a targeting ligand54.

Fig. 5
figure 5

Magnetic behavior (A), TGA (B), UV-Vis (C), and EDX (D, E, F, G, H, I, J, K) curves of NPs. (Description of colors and types of curves into figure).

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In vitro diagnostic analysis of NPs

In vitro cytotoxicity assay Investigation of the ability of FSM3, FHS2, FSMPF1 and FHSPF2 in interaction and protection of DNA against enzymatic digestion

With increasing concentrations and types of NPs, the surface charge of DNA is neutralized and the DNA in the well remains motionless. According to the results of agarose gel electrophoresis, all of the FHSPF and FSMPF have a higher neutralization ability than all PEI, FHS or FSM to neutralize the negative charge of DNA. As a result, the negative charge of DNA (5 g) was neutralized at 100 and 500 g concentrations of FSMPF1 and FHSPF2, respectively. Since FHSPF and FSMPF NPs have non-cationic polymers (PEG-FA), they may be less cationic than MNPs because they are coated with non-cationic polymers. HS and SM polymers also contain high cationic amine groups that interact electrostatically with negative charges of DNA’s phosphate groups and neutralize them. When high ratios of nano-vectors to DNA are present in the agarose gel, DNA movement becomes reduced towards the positive pole of the device. The observed absence of the PEI/DNA band in the retardation assay is likely due to excessive DNA condensation from PEI’s high positive charge, hindering gel migration. Large aggregate formation and potential DNA degradation at high PEI concentrations may also contribute. Sample preparation errors, such as improper ratios or mixing, represent further possible causes for this result. For this reason, higher concentrations of NPs of FSM1 are needed than those of FHSPF3 to neutralize the negative charge on DNA (Fig. 6, images A and B). In this study, NPs were investigated for their ability to protect DNA from restriction enzymes. Both in vivo and in vitro, restriction enzymes degrade DNA during gene transfer to mammalian cells. The cell’s defense mechanism destroys more than 99% of DNA transferred via the endocytosis-dependent pathway. The ability of NPs to protect DNA from restriction enzymes is therefore essential. Based on the results of this study, NPs are more effective at protecting DNA against DNase I when they are more concentrated. As can be seen in Fig. 6, image C and D, no bands of DNA were observed in the well of control DNA that had been treated with DNase I, indicating that it had been destroyed by the enzyme. When FHS or FSM NPs are blown to a concentration of 100–500 g, they cannot protect DNA against DNase I. When the concentration of NPs was increased up to 1000 g, the resolution of agarose gel bands gradually improved. NPs containing FSM/DNA did not degrade compared to the control at concentrations up to 1000 g. Furthermore, NPs containing FSMPF1 or FHSPF2 proved to be highly effective in preventing DNA degradation against DNase I (Fig. 6, images C and D)15,43,55,56.

MTT assay

In clinical gene therapy, cationic polymers are not used due to their high toxicity or unwanted interactions with macromolecules in the body. Polymers such as PEI, Folic acid, Spermine, etc., are very efficient at transferring genes to mammals; however, these compounds are not used due to their low efficiency in gene transfer to mammalian cells. A MTT assay was used to investigate the toxicity of PEI (control), FSM3, FHS2, FSMPF1 and FHSPF2. Based on the results, the FSMPF1 and FHSPF2 MNPs were more biocompatible with MCF-7 cells than the FSM3 and FHS2. After treating MCF-7 cells with 100 g/mL of PEI, FSM3, and FHS2, the lowest cell viability percentage was 42.3% (72 h), 84.9% (24 h), and 86.7% (24 h), respectively. In contrast, FSM3 NPs were significantly more toxic than FSMPF1 and FHSPF2. PEI treatment of MCF-7 cells at a 1000 g concentration (26.92% − 72 h) significantly reduced cell viability compared to FSMPF1 (84.22% − 72 h) and FHSPF2 (90.01% − 72 h) NPs (Fig. 6, images E-H)3,50,57,58.

DNA release pattern from FSM3/DNA, FHS2/DNA, FSMPF1/DNA and FHSPF2/DNA

Using two media of different pH and varying dextran sulfate concentrations (10, 20, 30, 40 and 50 mg/ml), we compared the stability of FSM3/DNA, FHS2/DNA, FSMPF1/DNA and FHSPF2/DNA MNPs with PEI/DNA complex. DNA release in all MNPs increased significantly at concentrations greater than 30 mg/ml of dextran sulfate at pH = 7.4; however, at all times, the PEI/DNA complex released more DNA than any other MNPs. During incubation at pH = 7.4 for one hour, DNA released from the PEI/DNA complex was 48%, while DNA released from FSM3/DNA, FHS2/DNA, FSMPF1/DNA, and FHSPF2/DNA MNPs was 37.58%, 41.26%, 17.79%, and 19.04%, respectively. A redundancy in competition between polyanion and DNA for binding to PEI polycation accounts for the low stability of PEI/DNA complex against polyanions such as dextran; On the other hand, non-ionic hydrophilic polymers such as Spermine and PEG reduce polyanion binding, which increases the stability of DNA-containing cation-containing MNPs against plasma anions at pH 7.4. The results also showed that decreasing the pH from 7.4 to 4.8 increased DNA release in all MNPs and PEI / DNA complexes. In a low pH environment, Spermine, Polyethylene glycol, and Folic acid layers are degraded. We conclude that in comparison to PEI in the physiological environment of the body, FSM3/DNA, FHS2/DNA, FSMPF1/DNA, and FHSPF2/DNA MNPs are significantly more able to protect DNA against premature release. Similarly, when DNA is released early in the endosome environment with a pH equal to 4.5 and the endosome binds to the lysosomes, and endolysosomes are formed, they increase DNA efficiency (Fig. 6, image I-L)26,50,58,59,60. The polymers FSMPF1 and FHSPF2 are continuous release materials. These MNPs release drugs in different ways depending on several factors, such as loading technique, molecular weight of polymer, characteristics of loaded material, etc. Previous studies have shown that using polymers with hydrophilic and hydrophobic components together increases DNA release rate32,61. DNA release rates have been improved by hydrophilic polymers’ ability to form water channels within matrixes. Hydrophilicity increases the rate of DNA release from FSMPF1/DNA and FHSPF2/DNA MNPs due to the binding of PEG, Hyperbranched Spermine, Spermine, and Folic acid. By changing each of the components of these MNPs’ molecular weights, it is also possible to modify the speed of DNA release according to the target.

Fig. 6
figure 6

Retardation assay of FSM3, FHS2, FSMPF1 and FHSPF2 at different concentration of MNPs (A and B); Agarose gel images of DNA extracted from FSM3, FHS2, FSMPF1 and FHSPF2 NPs after enzyme treatment (C and D); The cell viability after treatment with FSM3, FHS2, FSMPF1 and FHSPF2 NPs in 12 h (E), 24 h (F), 48 h (G), and 72 (H); and DNA release from PEI/DNA complex, FSM3/DNA, FHS2/DNA, FSMPF1/DNA and FHSPF2/DNA MNPs in environments containing pH = 7.4 (Upper) and pH:4.8 (Below), for half an hour (I), one hour (J), two hours (K), and three hours (L).

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Transfection of MCF-7 cells using PEI/DNA, FSM3/DNA, FHS2/DNA, FSMPF1/DNA, FHSPF2/DNA, and FHSPF3/DNA

During intracellular transfer and under cell transport, uncoated DNA is difficult to transfer into the cell due to reasons such as inadequate surface area and potential, early digestion by the cell defense mechanism, etc. A fluorescence microscopy and fluorescence apparatus demonstrated the ability of PEI/DNA, FSM3/DNA, FHS2/DNA, FSMPF1/DNA, and FHSPF2/DNA MNPs to deliver DNA to MCF-7 cells (Fig. 7, image A-N). MCF-7 cells were observed to emit green emission after treatment with MNPs such as FSM3/DNA, FHS2/DNA, FSMPF1/DNA, and FHSPF2/DNA. These MNPs were capable of transmitting and releasing DNA within MCF-7 cells; however, uncoated DNA was not able to pass into these cells. Based on flow cytometry, FSMPF1/DNA and FHSPF2/DNA MNPs displayed the highest gene transfer efficiency in serum (52% and 57%) and serum-containing medium (46% and 53%). In serum-containing and serum-free medium, PEI/DNA (31% and 14%) showed the lowest gene transfer efficiency, and FSM3/DNA (34% and 29%) showed the highest. Comparing PEI/DNA complexes with other MNPs, these results revealed a low level of gene transfer efficiency. MCF-7 cells can transfer genes to FHSPF MNPs in serum-containing medium, according to this study. With an outer shell made of PEG, Spermine, and Folic acid, these MNPs prevent nonspecific interactions with serum and minimize particle aggregation. Sterilizing particles also reduces the tendency to aggregate, thereby enhancing storage and application stability of formulations. It can be concluded that MNPs FSMPF1 and FHSPF2 is very effective when it comes to delivering DNA, because they protect DNA from degradation or damage. According to other researchers, controlled release of these complexes can result in sustained gene expression when released in controlled ways. These findings are indirectly confirmed by these evaluations58,62,63.

We also examined the function of the complex MNPs involved in DNA interaction by utilizing luciferase as a tool for determining level of gene promoter activator (EGFP-N1 gene expression in MCF-7 cells) in dealing with complexes. For comparison, MNPs with similar particle sizes and zeta potentials were used in this study since particle size and surface charge have a significant effect on gene transfer efficiency. MCF-7 cells were treated with two different RPMI media without or with 10% FBS to determine the efficiency of the MNPs in delivering DNA to them. In Fig. 7, you can see that MNPs FSMPF1 and FHSPF2 performed better in terms of percentage of positive cells (Fig. 7, image K) and Luciferase activity (Fig. 7, image N), which indicates that these MNPs are highly efficient at transfection. The MNPs FHSPF2 is very effective at delivering DNA due to its ability to protect it from degradation or damage. Other researchers have found that sustained gene expression can be achieved through controlled release by similar complexes. These conclusions are indirectly confirmed by these other researchers’ findings3,64,65. Figure 7 illustrates the ability of PEI / DNA, FSM3/DNA, FHS2/DNA, FSMPF1/DNA, and FHSPF2/DNA MNPs to penetrate and transfer into MCF-7 cells. DNA release from nano-vectors into cells is another important factor in the transfer of genes. MCF-7 cells can release DNA safely into these MNPs according to fluorescence microscopy images and fluorocytometric results (Fig. 7). Gene therapy research requires the protection of DNA against destructive enzymes since endocytosis is the basis for gene transfer with nano-vectors and more than 95% of DNA entering the cell is destroyed by intracellular nucleases. Researchers have found that poly-cations bind to DNA and thereby prevent restriction enzymes from binding to DNA, preventing DNA from being digested by them45. The FeO4, Spermine, PEG coating of our MNPs/DNA protects DNA against polyanions in the environment by preventing premature DNA release. According to them, poly-anions cannot penetrate Fe3O4-PEG due to the impermeable layer15. DNA is protected against enzymatic digestion by the interaction of FSM3, FHS2, FSMPF1, and FHSPF2 MNPs (Fig. 7). As a result, PEG, Hyperbranched Spermine, Spermine, and Folic acid reduce DNA contact surfaces and prevent restriction enzyme binding11,66.

Fig. 7
figure 7

Flow cytometry (A, B, C, D, I, and J) and fluorescence (E, F, G, H, L, and M) of MCF-7 cells; MCF-7 cells treated with PEI/DNA (A and E), siRNA-FAM (B and F), FSM3/siRNA-FAM (C and G), FHS2/siRNA-FAM (D and H), FSMPF1/siRNA-FAM (I and K), FHSPF2/siRNA-FAM (J and M) MNPs in serum-free medium containing FBS; Percentage of positive cells of the FSM3/siRNA-FAM, FHS2/siRNA-FAM, FSMPF1/siRNA-FAM and FHSPF2/siRNA-FAM NPs (K); and Luciferase activity of the FSM3/siRNA-FAM, FHS2/siRNA-FAM, FSMPF1/siRNA-FAM and FHSPF2/siRNA-FAM NPs (N).

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In vivo diagnostic analysis of NPs

In vivo toxicity analysis

Kaplan-Meier was used to analyze the survival data. In comparison to the PEI/siRNA-FAM complex, the mice in the FSMPF1/siRNA-FAM and FHSPF2/siRNA-FAM groups have a more favorable overall survival. As a result, the survival rate of control mice and FSMPF1/siRNA-FAM and FHSPF2/siRNA-FAM groups remained 80% up to 30 d, while the Kaplan–Meier curve showed a 50% decrease in survival rates when PEI/siRNA-FAM were used versus mice that were given FSMPF1/siRNA-FAM and FHSPF2/siRNA-FAM groups. Systemic toxicity was measured by body weight change (Fig. 8). Body weight changes were used to evaluate the safety profiles of PEI/siRNA-FAM, FSMPF1/siRNA-FAM, and FHSPF2/siRNA-FAM. The control mice were those without injection and those injected with PBS containing siRNA-FAM. Compared to mice injected with 50 g of siRNA-FAM in PBS, the mice without injection did not show a significant difference in body weight. Furthermore, the FSMPF1 and FHSPF2 groups did not significantly differ from the PBS group over the in vivo study period. Although mice treated with the FSMPF1 lost a small amount of weight (about 4.3%), it was not statistically significant as compared to mice treated with the PBS. The PEI/siRNA-FAM group showed the greatest decrease in body weight (17% compared to PBS). We found that siRNA-FAM delivered by the FSMPF1/siRNA-FAM and the FHSPF2/siRNA-FAM significantly improved efficacy and minimal toxicity43,45,61. Several gene-associated human diseases have been treated and diagnosed with targeted nanocarriers over the last few decades. However, NPs remain a major health concern due to their toxicity and ability to accumulate in healthy tissues as evidenced by studies indicating induced inflammation, DNA damage, and cellular dysfunction67,68. Therefore, a critical need exists for further research to develop safe and effective nanocarriers for clinical applications.

In vivo transfection assay

There were 17.34 and 76.12 and 184.99 ng/g tissue, siRNA-FAM concentrations in tumors extracted from mice that had been treated with siRNA-FAM, FSMPF1/siRNA-FAM, and FHSPF2/siRNA-FAM, respectively. According to Fig. 8, the results of the gene transfection assay using NPs in media containing FBS were in good agreement with the results of the in vitro transfection assay using the NPs. Polyethylene glycol is believed to be responsible for preventing non-specific interactions between MNPs NPs and serum compounds as described above. PEG imparts steric hindrance by creating a hydrated layer around nanoparticles, effectively preventing protein adsorption and aggregation. This mechanism enhances nanoparticle stability in biological media by minimizing non-specific interactions. Consequently, PEGylation significantly improves biocompatibility, reducing immunogenicity and prolonging circulation time. Understanding this effect is crucial for designing nanocarriers with enhanced therapeutic efficacy. Furthermore, as is well known, cancer cells express overexpressed folic acid receptors on their surface, which makes MNPs NPs particularly effective in protecting against cancer. Consequently, it seems that the presence of polyethylene glycol and folic acid on the surface of MNP NPs prevents macrophages from detecting them and also increases their absorption into cancer cells. The transfer of siRNA-FAM to tumor tissue was enhanced by external magnetic fields applied to the tumour site. In this study, FSMPF1/siRNA-FAM NPs transferred of RNA (from 76.12 to 281.27 ng/g tissue) to tumor tissue after applying an external magnetic field to the site, and FHSPF2/siRNA-FAM NPs transferred of RNA (from 184.99 to 391.38 ng/g tissue) to tumor tissue after applying an external magnetic field25. Diagnostic and therapeutic applications of MNPs have been successful. One of the most important topics investigated in recent years has been the application of MNPs to MRI imaging and hyperthermia (heat therapy). The magnetic properties of these MNPs also enable them to be delivered to target tissues11,15,43. Another problem with using nano-conductors in pharmacy is loading different drugs on their surfaces, in addition to all the benefits mentioned above18. It was demonstrated in this study that biodegradable MNPs and MNPs have a number of advantages. These findings led to experiments using these MNPs simultaneously in drug delivery systems, so that nano-carriers can be designed to make the most of the advantages of several nano-conductors at the same time while also covering their disadvantages. Many researchers have studied folic acid’s ability to treat cancer cells in recent years50. Compared to normal cells, most cancer cells contain significant amounts of folic acid receptors, which reduces their non-specific uptake into other cells when targeted with folic acid. The use of folic acid to target drug delivery to cancer cells is particularly attractive since it is absorbed by cancer cells through binding to folic acid receptors and endocytosis. The results of animal studies and flow cytometry demonstrated that folic acid-containing MNPs (FSMPF1 and FHSPF2) transport DNA to MCF-7 cells with higher efficiency than other MNPs used in this study in FBS-containing and FBS-depleted medium.

Fig. 8
figure 8

In vivo toxicity assays - Kaplan–Meier curves of BALB/c mice treated with different types of NPs (A); Changes in body weight and biochemical markers of BALB/c mice after treatment with different transfection reagents (B); The transfection efficiency of the NPs to tumor tissue in breast cancer-bearing BALB/c mice (C); And in vivo transfection by siRNA-FAM, FSMPF1/siRNA-FAM, and FHSPF2/siRNA-FAM NPs, Fluorescence images of tumor extracted from female BALB/c mice after treatment with FSMPF1/siRNA-FAM and FHSPF2/siRNA-FAM NPs NPs (F).

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Conclusion

Using cationic, magnetic, biodegradable, and dual-friendly polymers, this study proposes a multifunctional MNPs system for gene transfer. The present study showed that the simultaneous use of Fe3O4, PEG, Hyperbranched Spermine, Spermine, and Folic acid Co-polymers, along with reducing the toxicity (in terms of single use) and improving DNA release against poly-anions, also protected DNA from damage caused by enzymatic digestion. This study also demonstrated that FSMPF1/DNA and FHSPF2/DNA MNPs have a high ability to transfer genes into MCF-7 cells when compared to PEI/DNA complexes in serum-containing medium.