Fe 3 O 4 @MCM-41-Hydroxyapatite-Teriparatide nanocomposite: as a highly efficient drug delivery system for bone tissue

doi:10.21203/rs.3.rs-2218141/v1

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Fe 3 O 4 @MCM-41-Hydroxyapatite-Teriparatide nanocomposite: as a highly efficient drug delivery system for bone tissue

https://doi.org/10.21203/rs.3.rs-2218141/v1

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Objectives: A novel nanocomposite for targeted delivery of Teriparatide as an efficient inject dosage to improve drug-induced side effects, precautionary limitations, and frequent injection challenges.

Methods: Core-shell structure (Fe₃O₄@MCM-41) by the co-precipitation process and loading of Hydroxyapatite-Teriparatide on its surface were synthesized successfully. The HPLC analysis at in-vitro and in-vivo tests with and without an external magnetic field and MTT assay were performed.

Results: MNPs had a roughly 10 nm-sized particle and a spherical-like shape. The hydrodynamic size range was 360–440 nm, and the zeta potential ranged from -15–70 mV. The drug concentration reached 505 μg within 30 minutes of exposure to the magnetic field, and the coexistence of Teriparatide, HAP, and, Fe₃O₄-MNPs increased the proliferation of bone tissue stem cells.

Conclusion: Efficient biocompatible nanocomposite (Fe₃O₄@MCM-41/HAP-Terip) and magnetic field can improve the therapeutic effect of osteogenesis, and rebate drug limitations.

Biological sciences/Biochemistry

Biological sciences/Biological techniques

Biological sciences/Biotechnology

Health sciences/Diseases

Physical sciences/Chemistry

Physical sciences/Engineering

Physical sciences/Materials science

Drug Delivery Carriers

Magnetic Nanocomposite

Biodegradable

Organic-Inorganic Mesoporous

MCM-41

Bone Healing

Bone fractures, especially of the spine and long bones, often have a severe impact on the quality of life of patients and are associated with high mortality [1-4], due to the nature and time of treatment of bone fractures and influential factors of the weakness of drug treatments such as low bioavailability, side effects, etc. Despite the common use of autologous bone grafts, there is a need to find ways to repair and rebuild bone due to high costs - the unavailability of transplants, etc. Many beneficial therapeutic agents and innovative technologies for bone fractures are available to treat bone fractures and bone disorders such as osteoporosis. Studies in this field are growing in the world, including the use of 3D printers and the construction of bone scaffolding [5], the use of finite element method software in surgeries [6], and methods for improved bone tissue density in fractures [7, 8], different drug delivery techniques expressly transport medications to bone fractures, as well as improvements in all of these delivery systems when used in combination treatment [9]. However, because bones are dispersed throughout the body [10], the blood concentration of systemic therapeutic agents must be at the limit where it can exert pharmacological activity at the peripheral location. It frequently causes severe systemic side effects, resulting in a small toxic-therapeutic window for bone disease therapy; hence innovative drug delivery strategies are needed to prevent such difficulties. Drug Delivery Systems (DDS) [11, 12] are mainly classified into two types, i.e., sustained and targeted drug delivery systems [9]. DDS holds chemicals for a long time and releases them in response to external signals, due to their healing effects over a long period without overuse of the drug, which is exciting.

One of the drugs that help bones heal is parathyroid hormone analogs. Teriparatide (PTH (1–34)) is a manufactured form of natural parathyroid hormone in the body. Parathyroid hormone is recognized to be an endocrine regulator of phosphorus and calcium balance in the body and, at low concentrations, can cause anabolic responses in bones. This hormone has been extensively researched in several animal fracture models and found to improve the rate of callus mineralization, callus production, bone remodeling, and bone density. According to one research study, short-term daily Teriparatide treatment improves postoperative functional results and fracture healing in unstable intertrochanteric fractures [13].In osteoporosis disorder, Teriparatide stimulates bone formation and replaces old bone tissue with new tissue (Recombinant PTH). However, other drugs have antiresorptive effects and prevent bone destruction. While the only FDA-approved anabolic drugs for the treatment of postmenopausal osteoporosis are Teriparatide (PTH (1-34)) and Abaloparatide, a PTHrP (parathyroid hormone-related protein), [14, 15], Teriparatide is one of the systemic drugs whose disadvantages in using high doses are aggravated drug-induced side effects and the risk of osteosarcoma [16-19] and includes precautions for use in people with a history of kidney disease, hypercalcemia, and cardiovascular disease [20], and also the problems of intermittent use during the 2-year treatment period [21].

One of the types of calcium phosphates is hydroxyapatite which contains ions such as fluoride, sodium, and magnesium in the mineral part of the bone. Attention and research on this compound have increased significantly in medicine in the last two decades [22]. Unique properties such as osteoconductivity and high biocompatibility potential for this material as a material used in bone grafts [23], bone scaffolds, and load-bearing implants, the material used in implant coating, drug delivery, and it has dramatically increased the conductivity of other biological molecules. In addition, the low solubility of hydroxyapatite in natural environments, the ability to control porosity, specific surface area, and surface factors are other suitable properties that can increase the use of apatite as a drug carrier through injection or surgery [24]. Hydroxyapatite has been used in drug delivery to damaged bone and cancerous areas. The prolonged hydroxyapatite decomposition rate in neutral and play environments causes the drug to enter the body gradually and slowly [25]. All kinds of molecules and drugs except small hydrophilic molecules can be loaded on hydroxyapatite. Small hydrophilic components are washed with the surrounding water and separated from hydroxyapatite. By synthesizing multilayer structures, several types of drugs can be loaded on apatite [26]. In this case, the critical point is that hydroxyapatite's main application in drug delivery is related to the delivery of the drug to the damaged parts of the bone.

The use of nanoparticles as medicinal carriers has received a lot of interest in recent years. Because of their mesoscopic effect, tiny object effect, quantum size impact, and surface effect, nanostructures exhibit distinct physical and chemical characteristics compared to bulk or atomic components. Among nanoparticles, iron oxide nanoparticles are an example of a widely used nanomaterial; sixteen pure phases of iron oxides are known to date [27]. Among iron oxides, Fe₃O₄magnetite nanoparticles have received more attention than other iron oxides due to their superparamagnetic properties, low curie temperature, non-toxicity, and, biocompatibility. [27-31]. The nanoparticles may be steered to the target tissue using an external magnetic field and released there. Targeted medication delivery decreases the medicine's harmful effects on surrounding healthy tissues while lowering the drug's needed dose. Marycz et al. showed that to create new biomaterials for the treatment of bone illnesses associated with bone loss when the equilibrium between bone-forming and resorbing cells is altered, Fe₃O₄ nanoparticles and magnetic fields can be used [32]. Magnetite nanoparticles are treated with organic or inorganic coatings to improve their biocompatibility for usage in medication delivery [33].

To prevent clot and oxidization of magnetite nanoparticles, surrounding the nanoparticles with protective agents is essential. The stability of the colloidal suspension is due to the protective agents' balance between repulsive forces and gravity [34]. Coating magnetite nanoparticles may control the loading, delivery, and release of medicine with the proper combination of coatings. Furthermore, appropriate coatings can minimize nanoparticle toxicity and improve biocompatibility [35]. Silica coating is the most common way to protect the surface of magnetite nanoparticles, this type of coating is especially useful in the field of catalysis and drug delivery. Because of its biocompatibility, inertness, and chemical stability, the silica coating can avoid the aggregation and improve the characteristics of magnetic nanoparticles [12, 36-39]. Mesoporous silicates offer appealing features such as a large surface area (~1000 m². g⁻¹), large pore size (2–50 nm), narrow pore size distribution, thermal and mechanical stability, well-defined pore geometry, toxicological safety, and biocompatibility [12, 40]. Furthermore, the silica supports' surfaces may be chemically changed with different functional groups [41-43]. Many studies investigate MCM-41's potential as a medication carrier for medicines with low solubility [44-46]. Another significant research factor on these materials is developing and applying more straightforward and efficient synthetic methods. Majid Abdouss et al. developed a magnetic mesoporous biodegradable nanocomposite for a controlled-release drug delivery system [47]. Zhang et al. blended the synthesis of iron oxide nanoparticles with the building of mesoporous silica, which allowed the loading of Rhodamine on these nanocomposites [41]. Ahmed Olovo et al. showed that thiol-functionalized Fe₃O₄@MCM-41 magnetic nanoparticles were highly influential in stabilizing L-Asparaginase (L-ASNase) and improving its stability [36]. One-pot synthesis of iron oxide/silica of porous mesoporous silica has also been previously reported by Mueller et al. [48]. And also, in a study by SIMONA et al., the pH dependence of folic acid delivery from mesoporous structures was analyzed, and it was found that release is pH sensitive [49, 50].

Several innovative nanocomposite systems have recently been developed for cell-specific targeting of bone, increasing drug solubility, improving drug stability, and preventing drug degradation, allowing drugs to reach their target location without being eliminated in systemic circulation. For example, selenium can regulate the proliferation and differentiation of bone marrow stem cells (BMSc). A porous selenium nanomaterial (Se@SiO₂) enhances bone marrow mesenchyme stem cell migration, osteogenic differentiation, and fracture repair in rats. Porous Se@SiO₂ nanocomposite has therapeutic potential for bone tissue engineering [51]. Because of their superior biocompatibility, biodegradability, and bioactivity, composite materials comprising Nano-sized Bioactive Glass (NBG) particles and biopolymers have recently been created for bone tissue synthesis [52]. Due to their stimulating bone growth and chemically binding to neighboring bone tissue, NBG particles are an appealing bone repair material.

For fracture repair, bone marrow stem cells (BMSc), growth factors, parathyroid hormone, or synthetic chemicals, such as bisphosphonates (BPs) and calcium phosphate cement, hydrogels, and nanoparticles, are also used [9], but they have limits. The necessity for large dosages due to low bioavailability, for example, limits bisphosphonates as targeted medicines, and adverse effects include mandibular necrosis, ulceration, and musculoskeletal discomfort [53].

In this study, using the most effective materials in bone healing, the design and fabrication of drug-carrying nanocomposite with core-shell structures by the synthesis of Fe₃O₄M.N.P.s prepared using co-precipitation method and development of a mesoporous silica layer on the surface of MNPs (Fe₃O₄@MCM-41) by using CTAB (cetyltrimethylammonium bromide) as a template and TEOS (tetraethyl orthosilicate) as a precursor of silica, for the use and loading of hydroxyapatite-Teriparatide on it, to improve drug-induced systemic side effects, precautionary limitations of the drug, bioavailability, and drug half-lives, we provided specialized and targeted drug delivery in bone tissue repair. Then MTT assay test was employed by the mouse embryonic NIH3T3 fibroblast cell line and, the Saos-2 human osteosarcoma cell line. Finally, drug release was tested in-vitro and drug delivery was tested in-vivo.

2.1 Materials and Apparatus

All the chemicals, including tetraethyl orthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), FeCl₃∙6H₂O, FeCl₂∙4H₂O, NH₃, NaOH, NaF, HCl, are of analytic grade from Merck company. Pure deionized water produced by double RO technology (0.0 micro siemens) was used. Hydroxyapatite was purchased with high purity from Nic ceram co, and teriparatide was built under the brand name Cinnopar by the Cinnagen company. Various instrumentation approaches, including X-ray Diffractometry (XRD), Transmission Electron Microscopy (TEM), Fourier Transform Infrared (FTIR) spectroscopy, dynamic light scattering (DLS, Malvern Zetasizer ZS, Malvern, UK) used for measurements of estimate dispersion and particle size of the carrier, the MTT assay test was executed for the evaluation of cell cytotoxicity, Brunauer-Emmett-Teller (BET), and Vibrating Sample Magnetometry (VSM), were utilized to characterize the produced nanocomposite. A Shimadzu Fourier transforms infrared (FT-IR) spectrometer (Japan, 470) in the range of 400–4000 cm⁻¹ was used to assess the quality and functional groups of the produced nanoparticles. A PHILIPS transmission electron microscope was used to examine the nanoparticles' size and shape (TEM, CM10 HT 100 kV). A universal ultrasonic DSA100-SK2 was used for chemical mixing, a digital Jenway pH meter equipped with a combined glass-calomel electrode was used for solution pH adjustments, Christ lyophilization (Germany) was used for freeze-drying, and a strong magnet (Nd-Fe-B, 1 Tesla) was used for separation of a solid magnetic phase from solution.

2.2 Synthesis of magnetite nanoparticles Fe₃O₄

The Fe₃O₄ nanoparticles were prepared according to our previous work by the precipitated method [54]. To create the stock solution of ferrous and ferric chloride, 6.3 g FeCl₃∙6H₂O, 4.0 g FeCl₂∙4H₂O, and 1.7 mL HCl (12 mol L^-1) were dissolved in 50 mL of deionized water in a beaker. The solution was then degassed with argon gas and heated to 90 ˚C in a reactor. Simultaneously, 250 mL of a 1.5 mol L^-1 ammonia solution was progressively added to the solution while protected by argon gas and vigorously stirred (1000 rpm). During this procedure, the solution temperature was always held at 90 ˚C, and argon gas was continually pumped into the solution to avoid oxygen infiltration. The Fe₃O₄ nanoparticles precipitate dispersed by a tesla magnet was collected from the reaction solution after the reaction and washed four times with 500 mL doubly distilled water. Finally, 6.2 mg mL^-1 Fe₃O₄ M.N.P.s were obtained.

2.3 Synthesis of Fe₃O₄@MCM-41

Before coating with silica, it is necessary to select a low concentration of magnetic nanoparticles and expose it to ultrasound to form a standard core-shell structure. Otherwise, the polyunsaturated iron oxide will form inside the silica shell. After fresh synthesis, Fe₃O₄ M.N.P.s were immediately coated with MCM-41 to prevent oxidation (coating operation). For this purpose, Fe₃O₄ M.N.P.s was poured into a 250 mL balloon and made up to volume with distilled water, then 50 ml of the resulting mixture was separated inside Erlenmeyer, and 10 mL of TESO solution was added to it and stirred on a magnetic stirrer at 40 ˚C for one hour, then 0.9 g of NaOH and 0.19 g of NaF were added and stirred for another hour. Then 4 g of CTAB surfactant as template was added slowly and stirred for 2.5 hours. It was kept closed in all Erlenmeyer entry conditions. The combination was then processed hydrothermally in an autoclave for 48 hours to generate a core/shell magnetic nanocomposite. Finally, the surfactant template was removed by calcination in a furnace at 600 ˚C [39, 55-58].

2.4 Synthesis of Fe₃O₄@MCM-41-Hydroxyapatite

This operation was performed in 3 steps. In step 1, 10 mL of deionized water into a beaker, and its pH was regulated to 5 by HCL, then 0.7 g of Fe₃O₄@MCM-41 nanocomposite was added to it for one the clock was stirred at 40 ˚C. In step 2, to activate hydroxyapatite, it was added (to a weight ratio of 1 to 5, respectively HAP/Fe₃O₄@MCM-41) to 10 mL of deionized water has pH=8.5 (adjusted by NaOH) and stirred at 40 ˚C for one hour, then solid phase was filtered and dried. In step 3, the resulting activated HAP is added to the solution given from step1 slowly and gradually and stirred for 30 minutes at 40 ˚C by an ultrasonic device. And at the end, the precipitate was separated and dried.

2.5 Preparation of Fe₃O₄@MCM-41-Hydroxyapatite-Teriparatide

For this purpose, a drug pen containing Teriparatide (Cinnopar^®) was used, including 250 mcg/mL and 2.4 mL. Half of it was gently combined with 0.1 g of nanocomposite (Fe₃O₄@MCM-41-Hydroxyapatite), poured on a glass plate, and frozen. Then the resulting mixture was dried by the freeze-drying method.

2.6 In-vitro Process

To measure the release process of a drug-containing composite in simulated in vitro conditions (INVITRO Test), a solution containing 10 mL of water and 10 mL of ethanol at 37.5 °C with different preset pH = 7.3, 5.6, 8 in this case, 0.1 g of Fe₃O₄@MCM-41-Hydroxyapatite-Teriparatide was stirred for 2 hours in the prepared solutions on the stirrer heater. Finally, the solid phase was filtered, the liquid phase was provided for High-Performance Liquid Chromatography (HPLC), and drug release was determined.

3.1 Characterization of the synthesized magnetic nanocomposite

The magnetic nanocomposite (Fe₃O₄@MCM-41-HAP-Terip) was evaluated via FT-IR, TEM, and, SEM techniques. The Vibrating Sample Magnetometry (VSM) measurement was used to determine the type of magnetic order as well as to identify the possible interaction effects between nanoparticles because of the shape, size, and surface effects of the particles. X-ray Diffraction (XRD) analysis was used to identify the crystalline phase present in the materials and determine the minerals and polymers, composition, and physicochemical properties. To explore the size distribution of nanocomposites and investigation of colloidal stability, a (DLS) analysis was performed. To evaluate the stability of the electrostatic field and the sample dispersion, the zeta potential was measured.

3.1.1 Study of chemical bonding

The FT-IR spectra of Fe₃O₄ M.N.P.s, Fe₃O₄@MCM-41, Fe₃O₄@MCM-41/Hydroxyapatite, and, Fe₃O₄@MCM-41/Hydroxyapatite/Teriparatide as shown in Fig. 1, validate the chemical structure of the produced nanocomposites. Broad peaks at 3000–3500 cm^-1 were observed for the O–H stretching vibrations of surface -OH and sorbed water. All the mesoporous compounds exhibit the distinctive absorptions of MCM-41 structure at 475 cm^-1, 820 cm^-1, and 1089 cm^-1. These absorptions result from symmetric and asymmetric bending and stretching modes of Si-O-Si links [59].

The bands at wave numbers 563 and 503 cm^-1 related to Fe-O vibrations in tetrahedral and octahedral sites, respectively, while a strong peak at 1100 cm^-1 matched the Si–O stretching vibration in the amorphous silica shell, confirming the Fe₃O₄ as the magnetic core. Fe₃O₄@SiO₂, Fe₃O₄@SiO₂/HAP, and, HAP spectra comparisons show no discernible difference. Additionally, silicon dioxide, which was produced from TEOS to form the Fe-O-Si bond, and the presence of hydroxyl groups are present in the case of (Fe₃O₄@MCM-41/HAP/Terip). The spectra show the C–H stretching and bending vibrations of tri alkoxy organosilanes at 1400 cm^-1, with a strong peak at 2800 cm^-1 attributable to the amorphous silica's S–H stretching vibration shell. The FT-IR analysis revealed identical spectra in both nanocomposites (Fe₃O₄ and Fe₃O₄@MCM-41), confirming the manufactured nanocomposites.

In fig.1 (b), because of the Teriparatide present, a strong amide connection with the C = O group is located at 1492 cm^-1. The interaction between the N-H bending and the C-N stretching of the C-N-H group results in the 1435 cm^-1 stretching bond [21].

3.1.2 Study of crystalline structures

X-ray diffraction (XRD) patterns of mesoporous MNPs revealed peaks that could be indexed in both mesoporous structures and MNPs. According to the XRD pattern in figure 2, the results appear that the attractive crystal structure is unaltered and is significantly kept through the synthesis process. It has too been affirmed that the Fe₃O₄ nanoparticles are included within the entire test. At the same time, the refraction crest at 2θ = ~23 ˚ can be related to the SiO₂ amorphous Fe₃O₄@SiO₂. The Fe₃O₄-MNP patterns feature peaks with 2θ = 18-70, (18.24, 24.16, 30.09, 35.32, 36.96, 43.06, 53.40, 56.96, 62.51, and, 65.77), which are identical to those of pure magnetite with spherical structure, and their XRD is compatible with that of the spinal magnetite pattern described by the Joint Committee on Powder Diffraction Standards (JCPDS file No. 19-0629).

Figure 3. shows that each of the single-phase compounds Fe₃O₄ MNPs and hydroxyapatite was correctly synthesized following the reference codes 00-019-0629 and 01-074-0566, and the peaks that appeared in the two-phase compound (Fe₃O₄@MCM-41) are the confirmed model of reference code 00-051-1380.

3.1.3 Study of magnetic properties

Vibrating Sample Magnetometry (VSM) was performed to assess the magnetic properties of Fe₃O₄, Fe₃O₄@SiO₂, Fe₃O₄@SiO₂-HAP, and (Fe₃O₄@SiO₂/HAP/Terip), in-room temperature. As shown in figure 4, The value of saturated magnetism and superparamagnetic samples were investigated. Fe₃O₄ had the highest level of saturated magnetization value (Ms=9.53 emu/g) and other components listed respectively (Ms=7.77, 6.76, 6.26 emu/g). Due to the silica and hydroxyapatite shells that have been coated on Fe₃O₄ nanoparticles, saturation magnetism has likely diminished. The nonmagnetic (HAP/Teriparatide) moieties were loaded onto the surface of the magnetic support, which is a rational explanation for the final nanocomposite's lower MS value; however, it is important to note that the nanocomposite's actual MS value was still high enough to allow for magnetic separation with a standard magnet [60]. As a result, Fe₃O₄nanoparticle distance is increased, and interaction strength is decreased [12].

3.1.4Microstructural investigations

SEM and TEM micrographs of the produced mesoporous magnetite nanocomposite (MMNPs) are presented in Figs. 5 and 6. The transmission electron microscopy (TEM) was carried out in four-scale bars. The images show that a SiO₂ shell has coated Fe₃O₄ nanoparticles and that several core-shell sample structures can be separated honestly, indicating the achievement of the goal sample. The final target nanoparticles were found to have an organic shell with a brighter hue and an inorganic core (Fe₃O₄) with a more dense and black appearance [61]. Fe₃O₄-MNPs were found to be spherical in the TEM images is nearly 10 nm, which is in a considerable deal of agreement with the crystallite size determined by the Scherrer equation using the matching XRD pattern's (311) plane about 5-7 nm was approximated as the thickness of the shell. Images from TEM and SEM revealed the aggregation of numerous ultrafine particles with diameters of around 15 nm.

In morphograhical characteristics by SEM, the hydroxyapatite crystals were nonuniform in size but had a highly regular rod-like shape. In particular, HAP's crystal size ranged widely from the submicron to the micron levels, with a length between 400 nm and 3 μm and a width between 50-100 nm [62].

3.1.5 Zeta potential and Dynamic Light Scattering (DLS)

The zeta potential value of Fe₃O₄ MNPs coated with silica by Laser Doppler electrophoresis over a voltage range of around ±200 mV is shown in Fig. 7, that the nanocarrier's zeta potential ranged from -15 to 70 mV. It attests to the nanocarriers' excellent stability since the greater surface charge of the particles makes them more stable [63, 64]. The covalent functionalization procedure has been effective, based on the form. Although electrostatic forces are crucial to this process, hydrogen bonds also play a significant role [65].

Using Dynamic Light Scattering (DLS) equipment with a scattering angle of 176.1 (Malvern Nano Zetasizer ZS 90, UK), the sizes of the particles and their distribution were examined according to our previous work [66]. The stated diameter is a particle size (z-average) an intensity-weighted average of two observations with an estimated inaccuracy of no more than 2%. Figure 7 displays the particle size distribution of colloids. Results showed that this range was between 360 to 440 nm. This number determined by SEM is lower than the results of DLS because DLS evaluates the hydrodynamic diameter of colloid particles in water while SEM displays the diameter of dried particles.

3.2 In-vivo study

3.1.1 Methods

To investigate the efficiency of the synthesized nano drug in the body (in-vivo), rats were used. The research was done with approval id: IR.IAU.REC.1401.034 from the research ethics committee's certificate (Research Ethics Committees of Islamic Azad University-Central Tehran Branch) and, all experimental protocols were approved by licensing committee. We confirm that all methods were performed in accordance with the relevant guidelines and regulations. The reporting in the manuscript follows the recommendations in the ARRIVE guidelines. The animals were obtained from an animal house at Kermanshah University and were hosted under special pathogen-free conditions. All manipulations were performed by the European vertebrate protection convention ETS 123. The animals were kept at a temperature of 21 ℃ and a humidity of 50-60 %. Drinking water and normal food were provided freely. Experiments were performed using rats (with an average weight of 260 g). Ketamine/Xylazine as the anesthetic agent was used.

3.1.2 Evaluation of functional results of nano-drugs

To inject into two groups of 8 (each group consisted of 8 animals; 4 males and 4 females), first the mice were anesthetized and then 10 mg of the synthesized nano drug was injected into the first group through the left leg artery. At the same time, 40.000 IU of teriparatide was injected into the second group. Immediately after the injection, the animals of the first group were exposed to an external magnetic field (1 Tesla) from the right-hand area. To investigate the effect of the magnetic field on the conduction of nano-drugs, the amount of drug in the rat bone tissue was investigated in time intervals of 2, 5, 10, and 30 minutes as shown in Table 3-1. For this purpose, at the mentioned times, the bone tissue was removed from the animal's body and the concentration of the drug in it was analyzed using HPLC analytical technique in the presence of a control sample. Also, for confirmation, the above process was evaluated once again with a group of 16 without applying the magnetic field and the effect of direct medication. The concentration of teriparatide injection in both groups was 40.000 IU.

Interestingly, the results of the concentration of nano drugs and conventional drugs in bone tissue without the application of a magnetic field did not differ significantly, which shows the importance of the effect of the magnetic field.

Table 3-1. The release concentration of Teriparatide at 2, 5, 10, and 30 after the in-vitro test.

Time (minute)	2	5	10	30
Concentration in the bone tissue of the first group (μg)	107	190	378	505
Concentration in the bone tissue of the second group (μg)	5	15	24	69

3.1.3 Evaluation of teriparatide concentration in bone tissue

After the in-vivo test of the drug and applying the prescribed times, the animal was immediately killed and the bone of its right hand was separated and weighed, then the whole tissue was dried with a freeze dryer and ground into powder (using a water/ethanol solvent in a 1:1 ratio), drug extraction was done and analyzed by the HPLC method. The HPLC data are available in the supplementary file. It is worth mentioning that to investigate the effect and amount of nano drug trapping in vital tissues such as lungs and..., the injection was done in the artery, also the lung was washed and the amount of drug in it was also checked, which was traced and it confirmed the effectiveness of nano drug by the magnetic field.

3.3 Cytotoxicity Studies

The cytotoxicity of free teriparatide, teriparatide-loaded nanocomposite (Fe₃O₄@MCM-41/HAP/Teriparatide), Fe₃O₄ MNPs, Fe₃O₄@MCM-41, and Fe₃O₄@MCM-41/HAP was assessed using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide). To assess the cytotoxicity, the standard non-malignant mouse embryonic NIH3T3 fibroblast cell line and, the Saos-2 human osteosarcoma cell line was employed. According to the results presented in Figure 8. In the constant drug concentration, for both NIH3T3 and Saos-2 cell lines, Fe₃O₄ MNPs have shown a very slight toxicity trend within 24 and 48 hours. This issue was also observed for the Fe₃O₄@SiO₂ sample; in the case of core-shell combination with hydroxyapatite, due to the positive effect of hydroxyapatite [24], the incidence of toxicity has improved compared to the previous two samples. In the case of the single drug, the parathyroid hormone analog has caused the proliferation and differentiation of bone tissue stem cells, and the relative viability of the cell increased with the presence of teriparatide. And in the Fe₃O₄@SiO₂/HAP/Teriparatide sample, due to the synergistic effect of the positive impact of proliferation and differentiation of bone tissue cells by hydroxyapatite and teriparatide, not only no toxicity was observed, but this combination has caused biocompatibility.

The results obtained in this study were consistent with previous work that cytotoxicity analysis of free and PTH-loaded bilayer implants showed no cytotoxicity in the MC3T3-E1 cell line after 24 h [15].

Fractures and bone illnesses impact a high percentage of the population. While many protein-based medications are now approved, therapy with these drugs can have significant adverse effects and be very costly. Using a nanocomposite carrying the drug made in this study makes us have local drug delivery. The clinical condition and requirements determine whether systemic or local treatment is appropriate. A combination technique can also be employed, in which a localized depot is inserted at the moment of injury to speed up the early phases of healing, followed by systemic therapy to boost bone production and mineralization.

The combination of the properties of Fe₃O₄ MNPs and mesoporous materials opens up many opportunities for tailoring the material for a broad range of applications, including hydrophilic and hydrophobic drug-carrying therapies via magnetic targeting, as well as selective drug extraction from complex biological environments, with combination of high load and narrow pore size distribution. Based on further represented analyses, Fe₃O₄@MCM‐41-Hydroxyapatite-Teriparatide as an advanced biocompatible drug delivery system has been synthesized successfully. Studies have shown that these built-in drug delivery systems can modify the bio-distribution of treatments, improve bone formation's therapeutic effect, drug-induced systemic side effects, drug half-lives, and bioavailability. Another advantage of this method is that a high drug density can be loaded on the nanocomposite, and there is the ability to control long-term drug delivery.

Conflict of interest

The authors declare no competing interests.

ETHICAL STATEMENT:

Ethics approval and consent to participate:

Approval id: IR.IAU.REC.1401.034 from the research ethics committee's certificate

Consent for publication:

All authors have given their consent for publication.

Availability of data and materials:

Data and materials may be obtained via the corresponding author upon request

Competing interests:

Not applicable

Funding:

No fund received.

Authors' contributions:

Hamid Raza Hosseini: Data curation, frontiers research, resources, investigation, writing an original draft, laboratory syntheses, conducting required analyzes and tests, writing- review and editing, invivo test.

Majid Abdouss: Conceptualization, Validation, Investigation, Supervision.

Mostafa Golshekan: Methodology, Formal analysis, Invivo test.

Acknowledgements:

The authors would like to thank the technical staff of the laboratory of the chemistry department, Amirkabir University of Technology and Guilan university of medical science

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No competing interests reported.

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Fe 3 O 4 @MCM-41-Hydroxyapatite-Teriparatide nanocomposite: as a highly efficient drug delivery system for bone tissue

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimental

2.1 Materials and Apparatus

2.2 Synthesis of magnetite nanoparticles Fe₃O₄

2.3 Synthesis of Fe₃O₄@MCM-41

2.4 Synthesis of Fe₃O₄@MCM-41-Hydroxyapatite

2.5 Preparation of Fe₃O₄@MCM-41-Hydroxyapatite-Teriparatide

2.6 In-vitro Process

3. Results and Discussion

3.1 Characterization of the synthesized magnetic nanocomposite

3.1.1 Study of chemical bonding

3.1.2 Study of crystalline structures

3.1.3 Study of magnetic properties

3.2 In-vivo study

3.1.1 Methods

3.1.2 Evaluation of functional results of nano-drugs

3.3 Cytotoxicity Studies

4. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Fe 3 O 4 @MCM-41-Hydroxyapatite-Teriparatide nanocomposite: as a highly efficient drug delivery system for bone tissue

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimental

2.1 Materials and Apparatus

2.2 Synthesis of magnetite nanoparticles Fe3O4

2.3 Synthesis of Fe3O4@MCM-41

2.4 Synthesis of Fe3O4@MCM-41-Hydroxyapatite

2.5 Preparation of Fe3O4@MCM-41-Hydroxyapatite-Teriparatide

2.6 In-vitro Process

3. Results and Discussion

3.1 Characterization of the synthesized magnetic nanocomposite

3.1.1 Study of chemical bonding

3.1.2 Study of crystalline structures

3.1.3 Study of magnetic properties

3.2 In-vivo study

3.1.1 Methods

3.1.2 Evaluation of functional results of nano-drugs

3.3 Cytotoxicity Studies

4. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

2.2 Synthesis of magnetite nanoparticles Fe₃O₄

2.3 Synthesis of Fe₃O₄@MCM-41

2.4 Synthesis of Fe₃O₄@MCM-41-Hydroxyapatite

2.5 Preparation of Fe₃O₄@MCM-41-Hydroxyapatite-Teriparatide