Hematin nanoparticles and methods of making the same

Abstract

The application relates to the technical field of medicines, in particular to a hematin nanoparticle and a preparation method thereof. The method is based on a pH regulation system to prepare the hematin nanoparticle. The hematin nanoparticle has high solubility in water, simple composition of hematin nanoparticles formed by water, uniform particle size, good dispersity and good dispersion stability in water. The availability and application prospect of the hematin nanoparticle in a biological system are improved.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and in particular to a heme nanoparticle and its preparation method. Background Technology

[0002] Heme iron (also known as iron protoporphyrin IX) is a typical representative of iron porphyrin compounds in living organisms. It is widely found in hemoglobin, myoglobin, and various cytochromes, acting as a cofactor in key physiological processes such as oxygen transport, electron transport, enzyme catalysis, and cell signal transduction. In nutrition and clinical medicine, heme iron is an important iron supplement. It is directly and completely absorbed through the intestinal specific receptor (HCP1), with an absorption rate as high as 15%–35%, far exceeding that of traditional non-heme iron supplements (such as ferrous sulfate). Furthermore, it causes no gastrointestinal irritation and is widely used to improve iron deficiency anemia. In addition, heme iron possesses various pharmacological activities, including antioxidant, anti-inflammatory, and ferroptosis-inducing activities, and has broad application prospects in tumor treatment, inflammation regulation, and research on iron metabolism-related diseases. However, due to its highly hydrophobic porphyrin planar structure, heme molecules are prone to π-π stacking and aggregation, resulting in extremely low solubility in neutral aqueous solutions (approximately 0.1 μg / mL). This severely restricts its formulation and in vivo application, whether as an oral iron supplement or as an injectable or local delivery system for antitumor or anti-inflammatory drugs. This solubility issue significantly limits its bioavailability and therapeutic efficacy. Existing technologies typically employ strategies such as organic solvent dissolution, surfactant solubilization, or cyclodextrin inclusion to improve its dispersibility, but these methods generally suffer from poor formulation stability and residual organic solvents.

[0003] Therefore, developing a simple and efficient preparation method to prepare heme into nanoparticles with uniform particle size, good stability, and significantly improved solubility or permeability has important scientific value and application prospects. Summary of the Invention

[0004] Therefore, the purpose of this application is to overcome the shortcomings of the prior art and provide heme nanoparticles and their preparation method. This addresses the problems of traditional heme molecules easily agglomerating in water, having low solubility, and traditional heme nanoparticles exhibiting poor stability in water and leaving organic solvent residues.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] First, this application provides a method for preparing heme nanoparticles, comprising the following steps:

[0007] S1. Mix heme powder, water, and alkaline reagent, and vortex to dissolve, to obtain a dark green iron protoporphyrin IX salt solution;

[0008] S2. The high-iron protoporphyrin IX salt solution, water, and polymer stabilizer are mixed to obtain a mixed reaction system with a heme concentration of 200-400 μg / mL and a polymer stabilizer concentration of 0.25-0.6% w / v. Then, under stirring, a pH adjuster is slowly added dropwise to adjust the pH of the system to 2.5-3.5. The solution color is observed to change from dark green to reddish-brown, resulting in a heme nanoparticle suspension. The polymer stabilizer is PVP K30.

[0009] S3. Centrifuge the heme nanoparticle suspension, discard the supernatant, resuspend and wash the precipitate with ultrapure water until the filtrate is neutral, and obtain heme nanoparticles.

[0010] Preferably, in the mixed reaction system obtained in step S2, the concentration of heme is 200~300 μg / mL, and the mass concentration of PVPK30 is 0.45~0.55%w / v.

[0011] Preferably, in step S2, the polymer stabilizer is added as an aqueous solution of the polymer stabilizer.

[0012] Preferably, the heme concentration in the dark green ferric protoporphyrin IX salt solution is 1~2 mg / mL.

[0013] Preferably, the mass concentration of the polymer stabilizer in the aqueous solution is 1-3% w / v.

[0014] Preferably, the alkaline reagent is selected from sodium hydroxide, potassium hydroxide, or calcium hydroxide; the pH adjuster is selected from dilute hydrochloric acid.

[0015] Preferably, in step S1, the mass ratio of heme powder to alkaline reagent is 1~3:40.

[0016] Preferably, the centrifugation in step S3 is carried out in a refrigerated high-speed centrifuge, and the centrifugation conditions include a centrifuge speed of 15,000 to 16,000 rpm, a temperature of 4 to 10°C, and a time of 10 to 15 minutes.

[0017] Preferably, steps S1 to S3 are all performed under light-protected conditions.

[0018] In a second aspect, this application provides a heme nanoparticle obtained by the above preparation method.

[0019] Preferably, the heme nanoparticles are spherical particles.

[0020] Preferably, the hydrated particle size of the heme nanoparticles is 100~800 nm, and the polydispersity index (PDI) of the heme nanoparticles is ≤0.3. More preferably, the hydrated particle size of the heme nanoparticles is 100~200 nm.

[0021] Preferably, the polydispersity index (PDI) of the heme nanoparticles is 0.1 to 0.2.

[0022] Compared with existing technologies, this application has the following technical advantages:

[0023] During the pH adjustment process of the system, this application rapidly induces a supersaturated state in the Hemin solution, resulting in explosive nucleation of molecules. From a thermodynamic perspective, the newly generated hydrophobic nuclei, in order to minimize surface free energy, spontaneously shrink into spherical structures with the smallest specific surface area during self-assembly. Simultaneously, the lactam rings contained in the PVP K30 molecules in the system encapsulate the primary nuclei through a hydrogen bond network, providing an isotropic steric hindrance effect. This not only blocks the directional growth of Hemin crystals but also inhibits the aggregation caused by π-π stacking between nanoparticles, thereby stabilizing the heme nanoparticles (HNPs) in a thermodynamically stable spherical dispersion morphology.

[0024] Using the above method, the heme nanoparticles prepared in this application have a simple composition, and the resulting nanoparticles have uniform particle size and good dispersibility; effectively avoiding crystal directional growth and particle aggregation, they have high stability in water; at the same time, the heme nanoparticles significantly improve the solubility of Hemin, overcoming its poor water solubility, thereby enhancing its availability and application prospects in biological systems. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some implementation methods of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0026] Figure 1 The image shows the particle size distribution and TEM image of the heme nanoparticles prepared in Example 1. Figure 1 a is the particle size distribution diagram of heme nanoparticles. Figure 1 b is a TEM image of heme nanoparticles (scale bar 200 nm). Figure 1 c is a TEM image of heme nanoparticles (scale bar 100 nm).

[0027] Figure 2 The hydration particle size distribution curve and PDI change curve of the heme nanoparticles prepared in Example 1 under different storage time conditions are shown.

[0028] Figure 3 The figures show the external-visible and Fourier transform infrared spectral analysis results of the heme nanoparticles and Hemin active pharmaceutical ingredient prepared in Example 1. Figure 3 a represents the UV-Vis spectra of heme nanoparticles and Hemin active pharmaceutical ingredient. Figure 3 b is the ultraviolet absorption standard curve. Figure 3 c represents the infrared spectra of heme nanoparticles and Hemin active pharmaceutical ingredient.

[0029] Figure 4 The XRD patterns of the heme nanoparticles, Hemin active pharmaceutical ingredient, and PVP K30 prepared in Example 1 are shown.

[0030] Figure 5 The figure shows the effect of PVP K30 concentration and Hemin dosage on the particle size of heme nanoparticles. Figure 5 Figure a shows the effect of PVP K30 concentration on the hydrated particle size and polydispersity index (PDI) of heme nanoparticles. Figure 5 b represents the effect of Hemin dosage on the hydrated particle size and polydispersity index (PDI) of heme nanoparticles. Detailed Implementation

[0031] The embodiments described in this specification are merely for explaining this application and are not intended to limit this application.

[0032] For simplicity, this specification only explicitly discloses certain numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form a range not explicitly stated.

[0033] Those skilled in the art will understand that the order in which the steps are written in the various embodiments or examples does not imply a strict execution order and does not limit the implementation process in any way. The detailed execution order of each step should be determined by its function and possible internal logic. Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, but sequentially is preferred.

[0034] The present application is further illustrated below with reference to embodiments. It should be understood that these embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0035] Example 1: Preparation of heme nanoparticles.

[0036] (1) Preparation of Heme Salt Solution: Weigh an appropriate amount of Hemin powder, add ultrapure water and 1 mol / L NaOH solution, and vortex to dissolve completely, obtaining a homogeneous and clear dark green soluble sodium ferric protoporphyrin IX salt solution (Hemin salt solution). The mass concentration of heme in this Hemin salt solution is 1.1 mg / mL, and the concentration of NaOH is 40 mg / mL. Separately prepare a 1.83% w / v (i.e., 18.3 mg / mL) PVP K30 aqueous solution and a 1 mol / L HCl aqueous solution for later use.

[0037] (2) Nanoparticles: 1 mL of Hemin salt solution, 3 mL of ultrapure water and 1.5 mL of PVP K30 aqueous solution with a concentration of 1.83% w / v were added to a vial in sequence to obtain a mixed reaction system with a heme concentration of 200 μg / mL and a PVP K30 mass concentration of 0.50% w / v; the magnetic stirrer was set to a stirring speed of 1200 rpm, and 1 mol / L HCl aqueous solution was slowly added dropwise for about 2 min during continuous stirring. The solution color was observed to change from dark green to reddish brown. The pH value of the mixed system was measured to be 3 at this time, and HNPs nanoparticle coarse suspension was obtained.

[0038] (3) Separation and purification: A refrigerated high-speed centrifuge was set at 15,000 rpm and 4°C. The crude HNPs suspension was centrifuged under these conditions for 10 min. The supernatant was discarded, and the precipitate was resuspended in ultrapure water. This operation was repeated twice. The resulting dark brown precipitate (heme nanoparticles) was reconstituted with ultrapure water under 60W sonication to obtain a uniformly dispersed HNPs suspension (i.e., heme nanoparticle preparation) with a near-neutral pH (pH=7±0.2). All the above operations were performed in the dark.

[0039] Example 1: Characterization of the physicochemical properties of HNPs.

[0040] The main purpose of this experiment is to examine the physicochemical properties of the heme nanoparticles (HNPs) prepared in Example 1.

[0041] I. Particle size and morphology characterization

[0042] The particle size of the HNPs prepared in Example 1 was determined by DLS. Figure 1 a) The results showed that the hydrated particle size of HNPs was 127.6±1.1 nm, and the polydispersity index (PDI) was <0.2. Transmission electron microscopy (TEM) observation results ( Figure 1 b、 Figure 1 c) The HNP nanoparticles are spherical, uniform in size, and well-dispersed, consistent with the DLS results. The spherical formation mechanism of HNPs is as follows: During pH adjustment, the Hemin solution quickly reaches a supersaturated state, inducing explosive nucleation of Hemin molecules. From a thermodynamic perspective, to minimize the surface free energy, the newly generated hydrophobic nuclei tend to shrink into spherical structures with the smallest specific surface area during self-assembly. Simultaneously, the polymer stabilizer PVP K30 in the system contains abundant lactam rings, which can encapsulate the newly generated primary nuclei through a hydrogen bond network. This isotropic polymer encapsulation provides steric hindrance, preventing both the directional growth of Hemin crystals and the aggregation of nanoparticles due to π-π stacking, thus stabilizing HNPs in a thermodynamically stable spherical dispersion.

[0043] II. Physical Stability Test

[0044] The heme nanoparticles were stored at room temperature, and the hydrated particle size and PDI were measured on days 1, 2, 3, 5, and 7. Results ( Figure 2 The results showed that HNPs exhibited excellent colloidal stability over 7 consecutive days. The particle size and PDI change curves did not show significant fluctuations, and the hydrated particle size remained between 130 and 140 nm. The nanoparticles were well dispersed, indicating that HNPs prepared by pH-controlled nano-sizing process have excellent colloidal physical stability in pure water medium.

[0045] III. Ultraviolet-Visible Spectroscopy and Solubility Analysis

[0046] To clarify the structural integrity of the drug during the nano-sizing process and to explore the non-covalent interaction between the drug molecule and the polymer stabilizer, ultraviolet-visible spectroscopy (UV-Vis) and Fourier transform infrared spectroscopy (FT-IR) analyses were performed on the active pharmaceutical ingredient, the corresponding stabilizer, and the lyophilized nanoparticle powder.

[0047] The results are as follows Figure 3 As shown, where, Figure 3 a represents the UV-Vis spectra of HNPs and Hemin active pharmaceutical ingredients. Figure 3 b is the ultraviolet absorption standard curve. Figure 3c represents the infrared spectra of HNPs and Hemin active pharmaceutical ingredients. (Example:) Figure 3 As shown in Figure a, distinct heme iron characteristic absorption peaks appeared at 384 nm and 383 nm in the UV spectra of HNPs and Hemin, respectively, with weak absorption peaks in the 500-700 nm range. These characteristics are consistent with the typical absorption features of the heme chloride porphyrin ring in the strong UV band and weak visible light absorption band. This indicates that the core porphyrin ring structure of ferric heme chloride was not degraded or destroyed during the nano-sizing process. Furthermore, the characteristic peaks of HNPs showed a slight red shift due to the encapsulation of PVP K30. In addition, based on the established UV quantitative standard curve: y = 0.117x + 0.0770 (R... 2 =0.997), the concentration of heme iron in the HNPs suspension (i.e. heme nano-preparation) was measured to be 107.78 μg / mL, which is about 1100 times higher than the inherent solubility of the original drug in neutral pure water (0.1 μg / mL).

[0048] IV. Infrared Spectroscopy Analysis

[0049] Figure 3 c shows the infrared spectral characteristics of Hemin, PVP K30, and HNPs. The Hemin active pharmaceutical ingredient is located at 3436.51 cm⁻¹. -1 The stretching vibrations of the NH group of the porphyrin ring and the OH group of the propionic acid side chain are visible at 2916.06 cm⁻¹. -1 and 1700.85cm -1 The peaks at these locations represent typical CH vibration peaks and carboxyl C=O stretching vibration peaks, respectively. In the HNPs spectrum, the original peak at 1700.85 cm⁻¹ of Hemin can be observed. -1 The characteristic peak is at 1645.40 cm⁻¹ -1 The broad C=O absorption peak nearby is mainly due to the presence of a lactam ring structure in PVP K30, which gives it a very strong hydrogen bond acceptor property, affecting the carbonyl absorption band signal of Hemin. Meanwhile, at 3400 cm⁻¹... -1 The absorption peak in the region also broadened to 3444.19 cm⁻¹. -1 The above results indicate that a hydrogen bond network is formed between the proton donor (such as the -COOH of the propionic acid group) in the Hemin structure and the carbonyl oxygen of PVP K30, further confirming that Hemin and PVP K30 undergo close co-assembly during nanostructuring, thereby improving the hydrophobic aggregation tendency of ferric heme molecules.

[0050] V. X-ray Diffraction Analysis

[0051] To evaluate the impact of general nanoparticle preparation processes on the solid-state physical properties (crystal form and crystallinity) of drugs, X-ray powder diffraction was used to determine the lyophilized powders of the active pharmaceutical ingredient, polymer stabilizer, and their corresponding nanoparticles.

[0052] Figure 4 X-ray powder diffraction (XRD) patterns of HNPs, Hemin, and PVP K30 are shown. The results indicate that the Hemin active pharmaceutical ingredient exhibits multiple characteristic diffraction peaks of its original heme structure near diffraction angles of approximately 10° and 24°, while PVP K30, as an amorphous polymer, displays two broad amorphous diffraction peaks near 12° and 22°. In the diffraction pattern of HNPs, the original sharp characteristic crystalline peaks of Hemin disappear, and the entire diffraction curve transforms into a smooth baseline without any crystalline diffraction characteristics. This indicates that during the pH-controlled co-assembly process of "alkali dissolution and acid precipitation," the π-π stacking and hydrogen bond network between Hemin molecules are broken down, and the molecules lose their ability to form a lattice under the embedding of the PVP K30 framework, ultimately transforming the Hemin molecules into amorphous HNPs.

[0053] Example 2: Effect of PVP K30 concentration on heme nanoparticle size.

[0054] HNPs were prepared according to the procedure in Example 1. The mass concentration of heme in the Hemin salt solution was fixed at 2 mg / mL. The effect of PVP K30 concentration on the particle size of HNPs was investigated by changing the final concentration of PVP K30 in the reaction system. Specifically, the pH of the HNPs nanosuspension solution was measured to be 2.5–3.5 when HCl aqueous solution was added dropwise to adjust the pH of the system, changing the solution from dark green to reddish-brown. The pH of the purified HNPs suspension (i.e., the heme nanoparticle formulation) was 6.8–7.2.

[0055] Figure 5 Figure a illustrates the effect of PVP K30 concentration on the hydrated particle size and polydispersity index (PDI) of Hemin nanoparticles (HNPs). As shown in the figure, without the addition of PVP K30, Hemin crystals grow unrestricted in the acidic medium, eventually forming visible micron-sized precipitates (particle size > 1000 nm). When PVP K30 is added, the particle size initially decreases and then increases with increasing concentration. This may be due to the steric hindrance effect of a suitable amount of polymer adsorbed uniformly on the particle surface, while excessive PVP K30 leads to an abnormal increase in solution viscosity, causing non-specific aggregation between particles. When the PVP K30 concentration in the reaction system is 0.5% w / v, the hydrated particle size of the purified suspension is the smallest, approximately 500 nm.

[0056] Example 3: Effect of Hemin dosage on heme nanoparticle size.

[0057] HNPs were prepared according to the method in Example 1. The concentration of PVP K30 in the mixed reaction system was fixed at 0.5% w / v. The dosage of Hemin (i.e., the concentration of heme in the mixed reaction system) was changed to investigate the effect of dosage on the particle size of HNPs.

[0058] Figure 5 b demonstrates the effect of Hemin dosage on the hydrated particle size and polydispersity index (PDI) of HNPs. The results show that the particle size of HNPs continuously increases with the increase of the initial Hemin concentration. When the Hemin dosage is controlled at 200 μg / mL, the hydrated particle size is about 150 nm and the PDI is less than 0.2, with good dispersion uniformity, which is in line with the experimental expectations.

[0059] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing heme nanoparticles, characterized in that, Includes the following steps: S1. Mix heme powder, water, and alkaline reagent, and vortex to dissolve, to obtain a dark green iron protoporphyrin IX salt solution; S2. The high-iron protoporphyrin IX salt solution, water, and polymer stabilizer are mixed to obtain a mixed reaction system with a heme concentration of 200-400 μg / mL and a polymer stabilizer concentration of 0.25-0.6% w / v. Then, under stirring, a pH adjuster is slowly added dropwise to adjust the pH of the system to 2.5-3.

5. The solution color is observed to change from dark green to reddish-brown, resulting in a heme nanoparticle suspension. The polymer stabilizer is PVP K30. S3. Centrifuge the heme nanoparticle suspension, discard the supernatant, resuspend and wash the precipitate with ultrapure water until the filtrate is neutral, and obtain heme nanoparticles.

2. The preparation method according to claim 1, characterized in that, In the mixed reaction system obtained in step S2, the concentration of heme is 200~300μg / mL, and the mass concentration of PVP K30 is 0.45~0.55%w / v.

3. The preparation method according to claim 1, characterized in that, Steps S1 to S3 are all performed under light-protected conditions.

4. The preparation method according to claim 1, characterized in that, The alkaline reagent is selected from sodium hydroxide, potassium hydroxide, or calcium hydroxide; the pH adjuster is selected from dilute hydrochloric acid.

5. The preparation method according to claim 1, characterized in that, In step S2, the polymer stabilizer is added as an aqueous solution of the polymer stabilizer.

6. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of heme powder to alkaline reagent is 1~3:

40.

7. The preparation method according to claim 1, characterized in that, The centrifugation in step S3 is carried out in a refrigerated high-speed centrifuge. The centrifugation conditions include a centrifuge speed of 15,000 to 16,000 rpm, a temperature of 4 to 10°C, and a time of 10 to 15 minutes.

8. A heme nanoparticle, characterized in that, It is prepared according to any one of claims 1 to 7.

9. The heme nanoparticles according to claim 8, characterized in that, The heme nanoparticles are spherical particles.

10. The heme nanoparticles according to claim 9, characterized in that, The hydrated particle size of the heme nanoparticles is 100~800nm, and the polydispersity index of the heme nanoparticles is ≤0.3.

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