Formulation of Lecithin-Modified Calcium Phosphate Nanoparticles with Improved Cellular Uptake as a Carrier for Bisphosphonates and Method for Preparing the Same

Abstract

【Means for solving the problem】 The subject of the present invention is a formulation of calcium phosphate nanoparticles with enhanced cell uptake as a carrier for bisphosphonates and a method for preparing the same.

Description

Technical Field

[0001] The present invention relates to a formulation of nanoparticles of calcium phosphate containing hydroxyapatite, said nanoparticles being modified with lecithin containing phosphatidylcholine, said formulation having enhanced cell uptake and being a carrier for drugs from the group of bisphosphonates, such as sodium alendronate. The present invention also relates to a method for preparing such a formulation.

Background Art

[0002] Methods for preparing lecithin-modified hydroxyapatite nanoparticles are described in Patent No. 229015 and Patent Application P.434278. The former discloses a method for batch preparation of hydroxyapatite nanoparticles in the presence of lecithin, and lecithin has two functions: a means for controlling the size and shape of the particles and a means for enhancing biocompatibility. This patent indirectly shows the effect of lecithin modification on biocompatibility. The latter document discloses a reactor for continuous synthesis of lecithin-modified hydroxyapatite nanoparticles. Since hydroxyapatite nanoparticles are morphologically and chemically similar to the mineral part of bone, they can be used for treatment. Such applications relate to the regeneration of bone damage and defects, and the promotion of reconstruction and reformation of the mineral part of bone tissue.

[0003] Bisphosphonates have a hydrolytically resistant -C-P(O)-(OH) 2It is a group of compounds with two bases. These compounds show strong affinity with the mineral component of bone, apatite (Zhang, S., Gangal, G. and Uludag, H. Chem Soc Rev 36, 507 - 531 (2006)). Also, because they have the activity to control the bone remodeling process, they have become essential for the treatment of osteoporosis and bone cancer for decades. Among bisphosphonate drugs, there are alendronic acid and its sodium salt, sodium alendronate, but their bioavailability during oral administration is very low (less than 1%) (Porras, A.G., Holland, S.D. & Gertz, B.J. Clin Pharmacokinet 36, 315 - 328 (1999)). Furthermore, when administered orally, several side effects occur in the upper digestive tract, and the most serious among them is the increased risk of cancer development (Sun, K., Liu, J.M., Sun, H.X., Lu, N. & Ning, G. Osteoporosis Int 24, 279 - 286 (2013)). Therefore, it is necessary to propose an oral - suitable bisphosphonate formulation to improve the bioavailability of the drug and reduce side effects.

[0004] The preparation of a formulation containing hydroxyapatite nanoparticles and alendronate is known in the art due to its potential dual - therapeutic effect.

[0005] J. Neamtu et al., J Therm Anal Calorim, 2017, 127:1567-1582E shows a method for synthesizing alendronate-hydroxyapatite nanoparticles from calcium nitrate, diammonium hydrogen phosphate, and alendronate by chemical precipitation for use in biomaterials. Nanoparticle formulations consisting of alendronate were obtained in a batch reactor in two ways: 1) by adding an alendronate solution to a mixture in which hydroxyapatite nanoparticles had precipitated, and 2) by adding an alendronate solution to one of the reagents. This publication also describes the use of crystallographic, spectroscopic, and thermal methods to chemically and physically characterize the composites and determine the release profile of alendronate. No additional modifications that could improve the bioavailability of the obtained formulations were pointed out in this study.

[0006] E. Boanini et al., Biomaterials, 29, 2008, 790-796 describes a method for preparing hydroxyapatite nanoparticles containing alendronate and their in vitro interactions with osteoclasts and osteoblast-like cells. According to the tests performed, the presence of bisphosphonate in the nanocrystals inhibits the proliferation of osteoclasts. In particular, osteoblasts cultured in the presence of nanoparticles with a relatively high alendronate content showed increased alkaline phosphatase and type I collagen activity. Here too, the authors did not mention any additional modifications to the particles that could potentially improve the biological availability of the formulation.

[0007] R. Bosco et al., Applied Surface Science 328, 2015, 516-524 shows a method for coating titanium bone implants with alendronate-hydroxyapatite nanocrystals with an alendronate content of approximately 30 wt%. The coating was achieved by electrostatic spray deposition (ESD) or immersion in a dissolved bisphosphonate solution. As a result of in vitro tests, it was shown that the nHA ALE crystals have the activity to reduce the number of viable osteoclasts.

[0008] The use of compositions containing hydroxyapatite and alendronate has also been disclosed in a number of patent gazettes. CN108478872A describes a method for manufacturing PMMA bone cement, and further includes a hydroxyapatite-alendronate nanocomposite for use in filling bone defects. Patent publication WO2009035265 presents a method for synthesizing and using calcium phosphate as a microparticle-based system for oral administration of agents from the bisphosphonate group containing alendronate in the treatment of osteoporosis. The drug content of the disclosed formulation ranges from 1 to 50 wt% based on 100 wt% hydroxyapatite, and the synthesis is carried out by crystallization from a water-oil emulsion system.

[0009] U.S. Patent No. 8,158,153 discloses an oral dosage form based on nanoparticles (with a size not exceeding 2000 nm) having an active bisphosphonate cation, and the said formulation contains a penetration enhancer and a chelating agent. The disclosed formulation does not contain calcium phosphate containing hydroxyapatite. US6783772B1 discloses an oral composition in the form of a tablet containing a therapeutic amount of sodium alendronate for releasing sodium alendronate in the stomach and esophagus. This formulation consists of a compressed granule core of sodium alendronate embedded in a therapeutically inert sugar-based fibrous matrix. EP2548441B1 discloses a sustained-release formulation for intravenous administration containing bisphosphonate.

[0010] To ensure sufficient delivery of bisphosphonates containing alendronate, an active agent for the treatment of osteoporosis, various types of formulations have been published in the literature, but there is still a need for formulations that improve cellular uptake and enhance the therapeutic effect.

Summary of the Invention

Means for Solving the Problems

[0011] The subject of the present invention is a formulation of nanoparticles of calcium phosphate, preferably hydroxyapatite, said nanoparticles being modified with lecithin, preferably phosphatidylcholine, said formulation having enhanced cell uptake and being a carrier for bisphosphonates, the bisphosphonate being selected from the group consisting of bisphosphonate drugs approved for medical use, namely alendronate and zoledronate, the bisphosphonate being encapsulated in the calcium phosphate nanoparticles in an amount up to 40% by mass, and the size of the nanoparticles being less than 200 nm.

[0012] Preferably, alendronate is introduced into the formulation as sodium alendronate at a concentration in the range of 5 mM to 15 mM based on the volume of the reaction mixture.

[0013] Preferably, zoledronic acid is introduced into the formulation as zoledronic acid at a concentration of 5 mM based on the volume of the reaction mixture.

[0014] The subject of the present invention is also a method for obtaining calcium phosphate nanoparticles comprising the following steps: a) dissolving Ca(NO) 32 -4H 2 O in a lecithin solution, b) dissolving (NH 4 ) 2 HPO 4 in a bisphosphonate solution, adjusting the pH of the solution obtained from step a) and the solution obtained from step b) to a value of 10, mixing the obtained solutions in a reactor to obtain a suspension, then centrifuging the suspension to obtain a precipitate, washing 4 times with ultrapure water, centrifuging to purify the precipitate, drying the precipitate at 50 °C for 12 to 24 hours, pulverizing the precipitate in a ball mill at a speed of 150 rpm for 10 minutes, and mixing the solutions in the reactor to obtain a suspension is carried out in a continuous or batch reactor. Preferably, the reactor used in this method is a continuous reactor.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0016] Preferred embodiments of the present invention include, inter alia, a formulation of hydroxyapatite nanoparticles modified with lecithin, said formulation comprising a bisphosphonate, preferably alendronic acid or its sodium salt. Such a preferred formulation is obtained by a precipitation reaction in a flow reactor, in which up to about 500 mg of sodium alendronate is used, which is the maximum allowable content of sodium alendronate (water solubility: 10 mg / mL) in the reagent solution (50 mL) in the precipitation reaction. Furthermore, such a formulation is precipitated in the presence of lecithin (about 98% phosphatidylcholine), promoting the uptake of the formulation nanoparticles into cells, and as a result potentially enhancing the bioavailability of the formulation.

[0017] Hereinafter, the present invention will be described in detailed embodiments. However, the provided examples are not limiting.

Example

[0018] Example 1 - nHAp - LE - AL 5 mM 0.3 g of lecithin (phosphatidylcholine, Lipoid SPC - 3, Lipoid GmbH, Germany) was weighed and dissolved in 50 mL of ultrapure water with stirring at about 60 °C for about 30 minutes. Next, 5.904 g of calcium nitrate (V) tetrahydrate - Ca(NO 3 ) 2 ·4H 2 O was weighed and dissolved in the previously cooled lecithin solution. 162.5 mg of sodium alendronate (5 mM based on 100 mL of the reaction mixture) was weighed and dissolved in 50 mL of ultrapure water. Next, 1.981 g of diammonium hydrogen phosphate - (NH 4 ) 2 HPO 4It was weighed and dissolved in a sodium alendronate solution. Using aqueous ammonia, the pH of both reagents was set to 10. The prepared solution was mixed in a flow reactor at room temperature at a constant dosing rate of 500 mL / h. A Y-shaped flow reactor with an inlet flow path 50 mm in length and an outlet flow path 10 mm in length, a square flow path cross-section with dimensions of 1 mm × 1 mm was used. The suspension (100 mL) obtained in the receiving tank was centrifuged at a speed of 4500 rpm for 30 minutes. Then, the supernatant was decanted, and the remaining precipitate was rinsed with ultrapure water and purified 4 times by centrifugation (10 minutes, 4500 rpm). The final product was dried at 50 °C for about 12 hours and then ground in a ball mill at 150 rpm for 10 minutes. The obtained product was an off-white powder. This product was named nHAp-LE-AL 5 mM.

Example

[0019] Example 2 - nHAp-LE-AL 10 mM 0.3 g of lecithin (phosphatidylcholine, Lipoid SPC-3, Lipoid GmbH, Germany) was weighed and dissolved in 50 mL of ultrapure water with stirring at about 60 °C for about 30 minutes. Next, 5.904 g of calcium (V) nitrate tetrahydrate - Ca(NO 3 ) 2 -4H 2 O was weighed and dissolved in the previously cooled lecithin solution. 325.0 mg of sodium alendronate (10 mM based on 100 mL of the reaction mixture) was weighed and dissolved in 50 mL of ultrapure water. Next, 1.981 g of diammonium hydrogen phosphate - (NH 4 ) 2 HPO 4 was weighed and dissolved in the sodium alendronate solution. Using aqueous ammonia, the pH of both reagents was set to 10. The prepared solution was mixed in a flow reactor under the conditions described in Example 1. The suspension (100 mL) obtained in the receiving tank was purified, and the final product was prepared in the same manner as in Example 1. The obtained product was an off-white powder. This product was named with the initials of nHAp-LE-AL 10 mM.

Example

[0020] Example 3 - nHAp - LE - AL 15 mM 0.3 g of lecithin (phosphatidylcholine, Lipoid SPC - 3, Lipoid GmbH, Germany) was weighed and dissolved in 50 mL of ultrapure water with stirring at about 60 °C for about 30 minutes. Next, 5.904 g of calcium (V) nitrate tetrahydrate - Ca(NO 3 ) 2 -4H 2 O was weighed and dissolved in the previously cooled lecithin solution. 487.5 mg of sodium alendronate (15 mM based on 100 mL of the reaction mixture) was weighed and dissolved in 50 mL of ultrapure water. Next, 1.981 g of diammonium hydrogen phosphate - (NH 4 ) 2 HPO 4 was weighed and dissolved in the sodium alendronate solution. Ammonia water was used to set the pH of both reagents to 10. The prepared solutions were mixed in a flow reactor under the conditions described in Example 1. The suspension (100 mL) obtained in the receiving tank was purified, and the final product was prepared in the same manner as in Example 1. The obtained product was an off - white powder. This product was named taking the initials of nHAp - LE - AL 15 mM.

Example

[0021] Example 4 - nHAp - LE - ZL 5 mM 0.3 g of lecithin (phosphatidylcholine, Lipoid SPC - 3, Lipoid GmbH, Germany) was weighed and dissolved in 50 mL of ultrapure water with stirring at about 60 °C for about 30 minutes. Next, 5.904 g of calcium (V) nitrate tetrahydrate - Ca(NO 3 ) 2 -4H 2 O was weighed and dissolved in the previously cooled lecithin solution. 145.1 mg of zoledronic acid monohydrate (5 mM based on 100 mL of the reaction mixture) was weighed and dissolved in 50 mL of ultrapure water. Next, 1.981 g of diammonium hydrogen phosphate - (NH 4 ) 2 HPO 4It was weighed and dissolved in a zoledronic acid solution. Using aqueous ammonia, the pH of both reagents was set to 10. The prepared solution was mixed in a flow reactor under the conditions described in Example 1. The suspension (100 mL) obtained in the receiving tank was purified, and the final product was prepared in the same manner as in Example 1. The resulting product was an off-white powder. This product was named taking the initials of nHAp-LE-ZL 5 mM.

Example

[0022] Example 5 (comparative) - nHAp-AL 5 mM Calcium nitrate (V) tetrahydrate - Ca(NO 3 ) 2 -4H 2 O 5.904 g was weighed and dissolved in 50 mL of ultrapure water. 162.5 mg of sodium alendronate (5 mM based on 100 mL of the reaction mixture) was weighed and dissolved in 50 mL of ultrapure water. Next, 1.981 g of diammonium hydrogen phosphate - (NH 4 ) 2 HPO 4 was weighed and dissolved in the sodium alendronate solution. Using aqueous ammonia, the pH of both reagents was set to 10. The prepared solution was mixed in a flow reactor under the conditions described in Example 1. The suspension (100 mL) obtained in the receiving tank was purified, and the final product was prepared in the same manner as in Example 1. The resulting product was an off-white powder. This product was named taking the initials of nHAp-AL 5 mM.

Example

[0023] Example 6 (comparative) - nHAp-AL 10 mM 5.904 g of calcium nitrate (V) tetrahydrate - Ca(NO 3 ) 2 -4H 2 O was weighed and dissolved in 50 mL of ultrapure water. 325.0 mg of sodium alendronate (10 mM based on 100 mL of the reaction mixture) was weighed and dissolved in 50 mL of ultrapure water. Next, 1.981 g of diammonium hydrogen phosphate - (NH 4 ) 2 HPO4 It was weighed and dissolved in a sodium alendronate solution. Using aqueous ammonia, the pH of both reagents was set to 10. The prepared solution was mixed in a flow reactor under the conditions described in Example 1. The suspension (100 mL) obtained in the receiving tank was purified, and the final product was prepared in the same manner as in Example 1. The obtained product was an off-white powder. This product was named with the initials of nHAp-AL 10 mM.

Example

[0024] Example 7 (comparative) - nHAp-AL 15 mM 5.904 g of calcium nitrate (V) tetrahydrate - Ca(NO 3 ) 2 -4H 2 O was weighed and dissolved in 50 mL of ultrapure water. 487.5 mg of sodium alendronate (15 mM based on 100 mL of the reaction mixture) was weighed and dissolved in 50 mL of ultrapure water. Next, 1.981 g of diammonium hydrogen phosphate - (NH 4 ) 2 HPO 4 was weighed and dissolved in the sodium alendronate solution. Using aqueous ammonia, the pH of both reagents was set to 10. The prepared solution was mixed in a flow reactor under the conditions described in Example 1. The suspension (100 mL) obtained in the receiving tank was purified, and the final product was prepared in the same manner as in Example 1. The obtained product was an off-white powder. This product was named taking the initials of nHAp-AL 15 mM.

Example

[0025] Example 8 (comparative) - nHAp 5.904 g of calcium nitrate (V) tetrahydrate - Ca(NO 3 ) 2 4H 2 O was weighed and dissolved in 50 mL of ultrapure water. Next, 1.981 g of diammonium hydrogen phosphate - (NH 4 ) 2 HPO 4It was weighed and dissolved in 50 mL of ultrapure water. Ammonia water was used to set the pH of both reagents to 10. The prepared solutions were mixed in a flow reactor under the conditions described in Example 1. The suspension (100 mL) obtained in the receiving tank was purified, and the final product was prepared in the same manner as in Example 1. The obtained product was an off-white powder. This product was named nHAp by taking the initial letters.

Example

[0026] Example 9 (comparative) - nHAp-LE 0.3 g of lecithin (phosphatidylcholine, Lipoid SPC-3, Lipoid GmbH, Germany) was weighed and dissolved in 50 mL of ultrapure water with stirring at about 60 °C for about 30 minutes. Next, 5.904 g of calcium nitrate (V) tetrahydrate - Ca(NO 3 ) 2 -4H 2 O was weighed and dissolved in the previously cooled lecithin solution. Next, 1.981 g of diammonium hydrogen phosphate - (NH 4 ) 2 HPO 4 was weighed and dissolved in 50 mL of ultrapure water. Ammonia water was used to set the pH of both reagents to 10. The prepared solutions were mixed in a flow reactor under the conditions described in Example 1. The suspension (100 mL) obtained in the receiving tank was purified, and the final product was prepared in the same manner as in Example 1. The obtained product was an off-white powder. This product was named nHAp-LE by taking the initial letters.

Example

[0027] Example 10 - Scanning electron microscope The size of individual hydroxyapatite particles was determined from scanning electron microscope (SEM) images. Prior to imaging, a 10 nm gold palladium conductive layer was sputtered onto the hydroxyapatite sample. A Q150T (Quorum, UK) sputter coater was used and the SEM images were taken with a scanning electron microscope equipped with SU8230 (Hitachi, Japan). Photographs of the test particles are shown in Figure 1. Particles with alendronate added to the synthesis had a significantly increased single particle size compared to particles without this active substance.

Example

[0028] Example 11 - Measurement of Particle Size The SEM images were digitally analyzed to determine the particle size of hydroxyapatite. The analysis was performed using the ImageJ program (version 2.3.0 / 1.53f). For each sample tested, 100 independent particle size measurements were made. The mean values and standard deviations are shown in Table 1. A significant increase in particle size was observed when sodium alendronate was added to the synthesis.

Example

[0029] Example 12 - Measurement of Particle Size in Aqueous Suspension The particle size measurement in the aqueous suspension was performed using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK) device equipped with a red laser with a wavelength of 633 nm. The sample for analysis was prepared by dispersing hydroxyapatite powder in 10 mM KNO 3It was prepared as a 1% (w / v) suspension. The sample was further sonicated for about 10 minutes using an ultrasonic homogenizer UP100H (Hielscher Ultrasonics, Germany), then diluted 50-fold, and filtered using a syringe filter with a pore size of 0.45 μm to remove contaminants and dust. The measurement was repeated 5 times at a constant temperature of 25 °C. The results are shown in Table 1 as the mean value and standard deviation. The mean value was calculated from the number-weighted particle size distribution. The increase in the average particle diameter of the particles containing sodium alendronate compared to the particles without the active substance indicates the possibility of encapsulation of the active substance inside the particles and / or attachment of the active substance to the particle surface.

Example

[0030] Example 13 - Measurement of Zeta Potential The zeta potential measurement was performed using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK) apparatus equipped with a red laser with a wavelength of 633 nm. The sample for analysis was prepared by suspending hydroxyapatite powder in 10 mM KNO 3 as a 1% (w / v) suspension. The sample was further exposed to ultrasound for about 10 minutes using an ultrasonic homogenizer UP100H (Hielscher Ultrasonics, Germany) and diluted 50-fold. The measurement was performed 5 times at a constant temperature of 25 °C. The results are shown in Table 1 as the mean value and standard deviation. The change in the zeta potential of the tested particle surface is not important, and the measured values indicate that the hydroxyapatite powder composed of the obtained lecithin and sodium alendronate and the powder composed only of sodium alendronate tend to form aggregates in an aqueous environment. However, these aggregates can be effectively dispersed by methods known in the art.

Example

[0031] Example 14 - Thermogravimetric Analysis Thermogravimetric analysis (TGA) measurements were performed using a Mettler Toledo TGA / DSC3+ instrument at a heating rate of 10 °C / min in the temperature range of 30 to 1000 °C. The tests were carried out with a continuous air flow rate of 30 mL / min. Based on the obtained results, the content of sodium alendronate in the prepared formulation was determined using a correlation formula: - For hydroxyapatite not modified with lecithin:

Number

Number

[0032] is the lecithin content of the LE-hydroxyapatite nanoparticle powder expressed as a mass fraction.

[0033] The calculated sodium alendronate contents (mass %) of the blended powders of the examples (Examples 1 to 4) and comparative examples (Examples 5 to 9) according to the present invention are summarized in Table 1.

Table 1

Example

[0034] Example 1 Measurement of promotion of cell uptake of 5-hydroxyapatite nanoparticles To test the promotion of cell uptake of hydroxyapatite nanoparticles surface-modified with lecithin, MG63 human osteosarcoma cells (Sigma-Aldrich, Germany) were used. Before the experiment, the cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) culture medium supplemented with 10% v / v fetal bovine serum and antibiotics (100 U / mL penicillin, 100 mg / mL streptomycin). 10 5 cells / mL of the cell suspension was transferred onto the surface of a sterilized round coverslip placed in the wells of a 24-well plate. 1 mL of the cell suspension was introduced into each well. Two replicates were prepared for each hydroxyapatite powder tested (n = 2). Lecithin-modified hydroxyapatite (nHAp-LE) and pure hydroxyapatite (nHAp) powder samples were prepared for testing. The powder samples for testing were prepared by making a 1% (w / v) suspension of hydroxyapatite powder in phosphate-buffered saline (PBS, pH = 7.4). The samples were further sonicated for about 10 minutes using an ultrasonic homogenizer UP100H (Hielscher Ultrasonics, Germany), then diluted 50-fold, filtered using a syringe filter with a pore size of 0.45 μm to remove contaminants and dust, and further sterilized. After incubating osteoblasts with the nHAp-LE and nHAp nanoparticle suspensions for 4 hours, the suspensions were drained from the cells, and the cells were washed twice with sterile PBS. The cells were fixed using paraformaldehyde by a method known in the art. The coverslips on which the cells were cultured were transferred to wet microscope slides and observed using a Zeiss LSM880 (Zeiss, Germany) confocal laser scanning microscope. Photographs of the specimens were taken using transmitted light and fluorescence of hydroxyapatite nanoparticles excited by a laser with a wavelength of 488 nm. In this way, the uptake of nHAp-LE and nHAp nanoparticles by osteocytes was shown (Figure 2). In the tests shown here, lecithin-modified nanoparticles were more readily taken up by osteocytes.

Example

[0035] Example 16 - Measurement of the Release Rate of Alendronate from the Formulation 500 mg of nHAp-LE-AL 5 mM powder (174.0 mg of AL powder) and nHAp-AL 5 mM powder (135.2 mg of AL powder) were weighed and each was suspended in 10 mL of release medium. In this way, three samples (n = 3) of each test substance were prepared for each experiment. PBS at pH 7.4 (Experiment 1) and 5.0 (Experiment 2) was used as the release medium. An experiment to test the alendronate release from the formulation of hydroxyapatite nanoparticles according to the present invention was conducted for 30 days. During the experiment, the temperature of the release test container was maintained at 37 °C and the suspension was stirred. Samples were taken at regular intervals. Before sampling, the test suspension was centrifuged (10 minutes, 4500 rpm). After centrifugation, 0.5 mL of supernatant was taken from each release rate test container twice, and the amount of release medium taken was immediately replenished in the container. Next, each sample was mixed with 0.5 mL of a 0.4 mM FeCl 4 solution in 4M HClO 3 . The absorbance (wavelength 290 nm) of the mixture thus prepared was measured, and the concentration of alendronate was determined based on a previously prepared standard curve. The results were shown as the average mass (mg) of the released alendronate relative to the mass (0.5 g) ± SD (n = 3) of the formulation sample used in the test. The release of the active substance from the formulation was faster under the lower pH conditions tested, which correspond to the pH of the cell endosome.

Claims

**Claim 1** A formulation of nanoparticles of calcium phosphate, preferably hydroxyapatite, wherein the nanoparticles are modified with lecithin, preferably phosphatidylcholine, the formulation has enhanced cell uptake and is a carrier for bisphosphonates, the bisphosphonate is selected from the group of bisphosphonate pharmaceuticals approved for medical use, the group including alendronate and zoledronate, the bisphosphonate is encapsulated in the calcium phosphate nanoparticles up to 40% by mass, and the size of the nanoparticles is less than 200 nm, a formulation characterized by this. **Claim 2** The formulation according to claim 1, wherein the alendronate is introduced into the formulation as sodium alendronate at a concentration in the range of 5 mM to 15 mM based on the volume of the reaction mixture, a formulation characterized by this. **Claim 3** The formulation according to claim 1, wherein the zoledronate is introduced into the formulation as zoledronic acid at a concentration of 5 mM based on the volume of the reaction mixture, a formulation characterized by this. **Claim 4** In a method for obtaining the formulation of claim 1, a. Dissolving Ca(NO ３ ), ２ ·4H ２ O in a lecithin solution; b. (NH ４ ) ２ HPO ４ dissolving in a bisphosphonate solution, and c. Adjusting the pH of the solution obtained in step a. and the solution obtained in step b. to a value of 10; d. Mixing the solution of step c. in a reactor to obtain a suspension; e. Centrifuging the suspension of step d. to obtain a precipitate; f. Purifying the precipitate of step e. by washing 4 times with ultrapure water and centrifuging; g. Drying the precipitate of step f. at 50 °C for 12 to 24 hours; h. Grinding the precipitate of step g. in a ball mill at a rotation speed of 150 rpm for 10 minutes, including, the method, wherein the steps are carried out in a continuous or batch reactor. **Claim 5** The method according to claim 4, characterized in that the reactor is a continuous reactor.

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