Preparation method and composition of albumin-based diphosphate polyethylene glycol modified manganese dioxide methylene blue nano suspension
By preparing albumin-based polyethylene glycol diphosphate-modified manganese dioxide methylene blue nanosuspension, the problem of staining and imaging in gastrointestinal tumor localization methods has been solved, achieving accurate and durable tumor localization and imaging, and improving biocompatibility and safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-07
- Publication Date
- 2026-04-07
AI Technical Summary
Existing methods for locating gastrointestinal tumors cannot achieve precise and persistent staining and imaging, and suffer from problems such as haloing and inaccurate localization, making it difficult to meet the needs of preoperative planning and intraoperative localization of tumors after neoadjuvant chemoradiotherapy.
Manganese dioxide and methylene blue nanoparticles were prepared by biomineralization using albumin-based polyethylene glycol bisphosphate-modified manganese dioxide and methylene blue nanoparticles. Combined with DP-PEG modification and methylene blue loading, MB/(DP-PEG)-BSA@MnO2 nanoparticle suspension was formed, which has the functions of macroscopic staining and CT/MR dual-modality imaging.
It achieves precise labeling and imaging of tumor sites, with long-lasting labeling effect, high biocompatibility, avoidance of systemic toxicity, excellent imaging performance, and accurate localization, solving the problems of haze and inaccurate localization in existing methods.
Smart Images
Figure CN121796641A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical nanomaterials technology, and specifically relates to a method and composition for preparing albumin-based polyethylene glycol diphosphate modified manganese dioxide methylene blue nanosuspension. Background Technology
[0002] Currently, for patients with locally advanced rectal cancer (LARC), neoadjuvant chemoradiotherapy (nCRT) often results in tumor regression, upward migration, or even fragmentation of the primary tumor, making it difficult to accurately determine its original boundaries on preoperative MRI and intraoperative digital rectal examination. This leads to difficulties in "in situ resection" during surgery and increases the risk of positive surgical margins. While nano-carbon suspensions can be used clinically for marking before nCRT, providing a long-lasting black staining effect that meets the needs of long-term nCRT, they have inherent limitations: diffuse staining at the marked site affects the surgical field and the assessment of tumor margins; and they are only visible to the naked eye, failing to provide imaging information for preoperative planning and intraoperative imaging. Therefore, there is an urgent clinical need to develop a composite contrast agent that combines precise, long-lasting staining with imaging enhancement to meet the pressing need for precise localization of gastrointestinal tumors after nCRT.
[0003] Currently, commonly used methods for locating gastrointestinal tumors in clinical practice include: preoperative endoscopy, endoscopic titanium clips + X-ray, imaging examinations (such as CT 3D reconstruction, PET-CT), and endoscopic marking, etc. However, these methods still have many limitations, including at least the following aspects: Limitations of conventional staining agents: 1. Indian ink: It diffuses severely and easily contaminates the field of vision. After being injected into the serosal layer of the gastrointestinal tract, it causes a large inflammatory reaction and is prone to adhesion. Moreover, it has many post-injection complications and is prone to leakage into the abdominal cavity, causing peritonitis or intestinal obstruction.
[0004] 2. Nano-carbon suspension injection and indocyanine green have the following common limitations: after injection, the marked site is diffusely stained, which affects the surgical field and the judgment of the tumor edge. The staining agent is only visible to the naked eye and cannot be visualized on CT, MRI and other imaging. Moreover, it is greatly affected by the operator's skill level.
[0005] 3. Methylene blue (MB) injection: It is inexpensive and suitable for temporary marking; however, it has strong diffusion and can easily cause extensive smearing in the surgical field; it may cause local skin allergies and cannot be visualized on CT or MR imaging.
[0006] Limitations in imaging and endoscopic procedures: 1. Imaging examinations: can only determine the approximate location, are greatly affected by anatomical factors, and their accessibility and universality are generally limited.
[0007] 2. Intraoperative endoscopy: During the operation, it is easy to cause significant gas accumulation in the gastrointestinal cavity. The use of endoscope will reduce the operating space of laparoscopic surgery, and the process is complicated, which will prolong the operation time. In addition, the positioning is inaccurate for some patients (such as those with significant intestinal tortuosity and redundancy).
[0008] 3. Titanium clips: They are prone to falling off before surgery due to gastrointestinal peristalsis, and their metallic properties prevent them from being visualized under MRI. In addition, due to the high mobility of some intestinal segments, their positioning is not accurate.
[0009] Therefore, there is an urgent clinical need to develop a combined functional contrast agent that combines accurate staining and imaging to achieve precise localization of gastrointestinal tumors. Summary of the Invention
[0010] This invention provides a method and composition for preparing albumin-based polyethylene glycol diphosphate modified manganese dioxide methylene blue nanosuspension, which has stable physicochemical properties, high biocompatibility, and functions as both visual staining and CT / MR dual-modal imaging.
[0011] This invention includes the following steps: S1: Preparation of BSA@MnO2: KMnO4 solution was slowly added to MnCl2·4H2O solution to form MnO2 precursor solution, and then the pH of MnO2 precursor solution was adjusted to 6; the pH-adjusted MnO2 precursor solution was subjected to ultrafiltration to separate MnO2 precipitate. The MnO2 precipitate was then washed and dried. The treated MnO2 precipitate was dispersed in pure water to prepare an MnO2 solution. The MnO2 solution and BSA solution were mixed at a volume ratio of 1:5 to form a BSA@MnO2 mixed solution. The BSA@MnO2 mixed solution was subjected to water bath ultrasonic treatment to obtain a stable BSA@MnO2 nanoparticle suspension dispersed in the liquid phase. S2: DP-PEG modification treatment: DP-PEG solid powder was dissolved in pure water and subjected to ultrafiltration and centrifugation to obtain DP-PEG solution. Then, LDP-PEG solution was slowly added to the prepared BSA@MnO2 nano suspension and stirred for 4 hours to obtain (DP-PEG)-BSA@MnO2 transition solution. The (DP-PEG)-BSA@MnO2 transition solution was then subjected to ultrafiltration and centrifugation to obtain the (DP-PEG)-BSA@MnO2 solution. S3: Methylene blue loading treatment: Methylene blue powder is dissolved in pure water and subjected to ultrafiltration and centrifugation to obtain an aqueous solution of methylene blue; The pH of the (DP-PEG)-BSA@MnO2 solution was adjusted to 7-8.5 to make its surface negatively charged, and the pH of the methylene blue aqueous solution was adjusted to 7 to make its surface positively charged. A certain volume of methylene blue solution and (DP-PEG)-BSA@MnO2 solution were mixed to ensure that the mass ratio of methylene blue to BSA@MnO2 in the mixed solution was maintained at 1:3. Finally, the (DP-PEG)-BSA@MnO2 solution and the methylene blue aqueous solution were mixed at a mass ratio of 1:3 and a concentration ratio of 3:1 and stirred continuously for 6 hours to obtain MB / (DP-PEG)-BSA@MnO2 nano-suspension.
[0012] The specific method for calculating the above solution is as follows: Calculate the total mass of BSA@MnO2 in the system as M(BSA@MnO2) = C × V [mg]. Based on a MB to BSA@MnO2 mass ratio of 1:3, the required MB mass is M(MB) = M(BSA@MnO2) / 3 [mg]. Calculate and measure the corresponding volume V(MB) = M(MB) / 1 [mL] of the prepared 1 mg / mL methylene blue stock solution, and add it to the (DP-PEG)-BSA@MnO2 solution with adjusted pH.
[0013] Furthermore, in step S1, the concentration of the KMnO4 solution is 4 mg / mL, and the concentration of the MnCl2·4H2O solution is 4 mg / mL.
[0014] Furthermore, in step S1, the concentration of the MnO2 solution is 4 mg / mL, and the concentration of the BSA solution is 20 mg / mL.
[0015] Furthermore, the concentration of the DP-PEG solution in step S2 is 5 mg / mL.
[0016] Furthermore, the volume of the LDP-PEG solution in step S2 is 160 μL.
[0017] Furthermore, the ultrafiltration operation in step S2 is performed at least twice.
[0018] Furthermore, in step S2, the centrifugation operation is performed at least three times, with a centrifugation speed of 400 r / min and a duration of 10 min for each centrifugation operation.
[0019] Furthermore, in step S3, the concentration of the methylene blue aqueous solution is 1 mg / mL.
[0020] A protein-based polyethylene glycol diphosphate-modified manganese dioxide methylene blue nanosuspension composition is prepared by the above-described method.
[0021] The beneficial effects of this invention are as follows: 1. This invention integrates the staining function of methylene blue with the CT / MR imaging function of manganese metal for the first time, and aims to construct an MB / (DP-PEG)-BSA@MnO2 nanosuspension with BSA as the carrier, and innovatively uses gastrointestinal serosa injection, while specifically responding to the tumor microenvironment; 2. This invention is the first to integrate the staining function of methylene blue with the CT / MR imaging function of manganese-based nanomaterials; after injection, the marked site is visible to the naked eye and can be clearly visualized under CT and MR, solving the problem that existing staining agents cannot perform imaging navigation, and realizing the simultaneous performance of staining and dual-modal imaging. 3. Strong biocompatibility and safety: Using BSA as the matrix, an appropriate amount of DP-PEG is added for modification and cross-linking to form (DP-PEG)-BSA@MnO2, which has high biocompatibility and low cytotoxicity. 4. Convenient administration route: It uses gastrointestinal serosal layer injection instead of intravenous injection, and its particle size is 70-100nm. Therefore, it avoids the systemic toxicity problems that may be caused by nanomedicines entering the blood through capillaries due to their small particle size (<50nm), which significantly improves the safety of clinical application. 5. High responsiveness to the tumor microenvironment: MnO2 can be reduced to Mn in the tumor microenvironment (pH<7, high glutathione GSH). 2+ (Based on the redox principle: MnO2 + 2GSH + 2H+ → Mn2++ + 2GSSG + 2H2O), Mn 2+ It is not only a highly efficient MRI T1-weighted contrast agent, but also a high atomic number element in CT imaging, enabling specific and efficient release and imaging at the tumor site; 6. Long-lasting and precise marking effect: By optimizing the formulation process, this invention aims to extend the staining and marking time, striving to surpass the staining time of simple MB injection solution (approximately 2-3 days) and titanium clip marking time (approximately 7 days). Furthermore, compared to nano-carbon suspensions, the staining diameter of this invention's product is smaller, resulting in more precise positioning.
[0022] 7. Through systematic experiments comparing various PEG modification groups, this invention ultimately selected DP-PEG-COOH as the preferred option. Through the strong anchoring effect of the DP group and the hydrophilic protection of the COOH group, a stable "anchoring-protection" structure is formed, ensuring the excellent long-term stability, high imaging performance and good biocompatibility of the nano-suspension. This selection is based on detailed experimental data and breaks through the limitations of the simple albumin-manganese dioxide system. Attached Figure Description
[0023] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a flowchart of the preparation technology route of the present invention; Figure 2 It is observed by the naked eye of MDPBMNs; Figure 3 These are the TEM results for MDPBMNs; Figure 4 These are the mapping analysis results of MDPBMNs; Figure 5a These are the AFM results for MDPBMNs; Figure 5b This is a three-dimensional topographic image of the AFM results of MDPBMNs; Figure 6 These are the XRD results of MDPBMNs; Figure 7 These are the FTIR results for MDPBMNs; Figure 8 This is the XPS total spectrum of MDPBMNs; Figure 9 This is the XPSC elemental spectrum of MDPBMNs; Figure 10 It is the XPSO elemental spectrum of MDPBMNs; Figure 11 It is the XPSMn elemental spectrum of MDPBMNs; Figure 12 It is the hydrated particle size of (DP-PEG)-BSA@MnO2; Figure 13 It is the hydrated particle size of MDPBMNs. Detailed Implementation
[0024] To make the objectives, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Several embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of the present invention will be thorough and complete.
[0025] like Figure 1As shown, an albumin-based nanosuspension is specifically a preparation of an albumin-based polyethylene glycol diphosphate-modified manganese dioxide methylene blue nanosuspension. Its structure is as follows: using bovine serum albumin (BSA) as a template and stabilizer, manganese dioxide (MnO2) nanoparticles are generated via biomineralization to form a BSA@MnO2 core; subsequently, the core is surface-modified with polyethylene glycol diphosphate (DP-PEG) to obtain (DP-PEG)-BSA@MnO2; finally, positively charged methylene blue (MB) is loaded onto the negatively charged (DP-PEG)-BSA@MnO2 using electrostatic adsorption, forming the final MB / (DP-PEG)-BSA@MnO2 nanosuspension.
[0026] I. Its key preparation steps include: 1. Preparation of BSA@MnO2: KMnO4 solution was slowly added to MnCl2·4H2O solution to adjust the pH of the solution. The solution was then subjected to ultrafiltration at room temperature to separate the MnO2 precipitate, which was then washed and dried. MnO2 was dispersed in water and mixed with BSA solution in a specific ratio. The mixture was then ultrasonically heated in a water bath to form BSA@MnO2 nanoparticles.
[0027] 2. DP-PEG Modification: This invention systematically compared the effects of different PEG modification groups (including NH2-PEG-NH2, CH3O-PEG-CHO, SH-PEG-SH, and DP-PEG-COOH) on the performance of nanosuspensions. It was found that DP-PEG-COOH modification significantly improved the stability, imaging performance, and biocompatibility of nanoparticles. Specifically, DP-PEG-COOH-modified nanoparticles exhibited the smallest hydrated particle size (83.2±2.1 nm), the narrowest particle size distribution (PDI 0.16±0.01), the highest absolute Zeta potential (-27.4±2.8 mV), and the longest suspension stability time (>30 days). Furthermore, in in vitro CT and MRI imaging, the DP-PEG-COOH-modified nanosuspension showed the highest CT value (446±23.3 HU) and relaxation rate (T1: 12.3±1.55 [Mn]mMs). -1 T2: 19.13 ± 1.88 [Mn]mMs -1 The cells exhibited good survival rates (79±2%). Therefore, DP-PEG-COOH is preferred as the surface modifier in this invention. The strong coordination of DP-PEG with BSA@MnO2 enhances the water solubility and biocompatibility of the nanoparticles, reducing the protein crown effect. The coordination binding of DP with MnO2 nanoparticles improves particle suspension stability. 3. MB loading: After adjusting the pH and MB content and removing impurities by ultrafiltration and centrifugation, MB / (DP-PEG)-BSA@MnO2 nano-suspension with a particle size of approximately 70-100 nm is obtained by physically mixing with positively charged MB using the principle of charge adsorption and continuous stirring. The finished product is stored at room temperature (4℃).
[0028] The above preparation method provides a novel nanosuspension with stable physicochemical properties, high biocompatibility, and dual-modal imaging capabilities of macroscopic staining and CT / MR – MB / (DP-PEG)-BSA@MnO2 nanosuspension.
[0029] II. The following are key studies on the properties of the prepared MB / (DP-PEG)-BSA@MnO2 nanosuspension: 1. Surface modification optimization research
[0030] To optimize the performance of nanosuspensions, this invention systematically evaluated the effects of four different PEG modification groups (NH2-PEG-NH2, CH3O-PEG-CHO, SH-PEG-SH, and DP-PEG-COOH) on the physicochemical properties, stability, development performance, and biocompatibility of nanoparticles.
[0031] Experimental conclusions and results analysis: NH2-PEG-NH2 (Dual Amino) Experimental Results: This scheme performed poorly. The nanoparticle size was relatively large (183.3±5.2 nm), and the Zeta potential was -14.6±1.6 mV. The system stability was significantly insufficient, with a suspension stability period of less than 7 days. Its imaging performance was poor, with low CT values (280±18.4 HU) and low relaxation rates (T1: 7.42±1.62, T2: 10.27±1.53 mMs). -1 The results were all low. The cell viability (81.23±2.24%) was acceptable.
[0032] The results suggest that the poor performance of diamino PEG is primarily due to its molecular structure. Although the measured Zeta potential is negative, the positively charged sites provided by the diamino groups may still interact strongly with negatively charged cell membrane components and serum proteins (such as albumin), leading to rapid protein adsorption (protein crown formation), cytotoxicity, and interparticle cross-linking and aggregation. More importantly, the bifunctional amino groups increase the risk of intermolecular cross-linking, resulting in decreased particle aggregation stability. This structural characteristic makes it less effective in maintaining the long-term stability of nanoparticles and also affects its imaging properties.
[0033] CH3O-PEG-CHO (Methoxy-aldehyde group) Experimental Results: The results of this method were moderate. The nanoparticle size was moderate (110.7±3.3 nm), the Zeta potential was -19.4±1.9 mV, and the dispersibility was acceptable (PDI 0.22±0.03). Suspension stability was improved (approximately 15 days), and imaging performance was moderate (CT value 320±20.8 HU, T1: 8.38±1.17, T2: 11.06±1.65 mMs). -1 However, the cell survival rate was relatively low (70±3%).
[0034] The results suggest that the reactivity of the aldehyde group (-CHO) is a key factor affecting its performance. The aldehyde group can form a Schiff base with an amino group, but this connection is unstable in an aqueous environment and easily hydrolyzes, leading to the shedding or recombination of the PEG layer. This instability directly affects the long-term stability of the nanoparticles and may also affect their biocompatibility. Incompletely reacted residual aldehyde groups or unstable nanostructures may introduce additional cytotoxic risks, resulting in relatively low cell survival rates.
[0035] SH-PEG-SH (bis-mercapto) Experimental results: This method performed extremely poorly. Although the initial particle size was small (102.8±3.6 nm), the system was extremely unstable. The Zeta potential could not be measured normally due to aggregation, and the suspension stability time was less than 0.5 days. Imaging performance was severely impaired (CT value only 103±25.7 HU, extremely low relaxation rate), and cytotoxicity was significant (cell viability 55±4%).
[0036] The results suggest that the failure of dithiol PEG is primarily due to the oxidative cross-linking properties of the thiol group. In an aqueous environment, the thiol group is readily oxidized to form disulfide bonds, leading to irreversible cross-linking and aggregation between nanoparticles. This intense aggregation completely destroys the colloidal stability of the nanoparticle formulation, causing it to precipitate rapidly. It also severely affects its imaging properties and biocompatibility. The aggregates may cause severe cell damage through physical blockage, triggering strong inflammatory responses, or oxidative stress.
[0037] DP-PEG-COOH Experimental Results: This scheme exhibits the best performance. The nanoparticles have the smallest particle size (83.2±2.1 nm) and the narrowest distribution (PDI 0.16±0.01), and the largest absolute value of the Zeta potential (-27.4±2.8 mV). The system demonstrates excellent long-term stability (sustainable suspension for approximately 30 days). Its imaging performance is significantly superior to other schemes, with lower CT values (446±23.3 HU) and lower relaxation rates (T1: 12.32±1.55, T2: 19.13±1.88 mMs). -1All parameters reached their highest levels. Cell viability was good (78.89±2.26%).
[0038] The results suggest that the superior performance of DP-PEG-COOH stems from its unique molecular structure design. DP (distearate), as a strongly hydrophobic anchoring group, can firmly embed itself into the hydrophobic regions of nanoparticles, providing stable membrane anchoring. COOH (carboxyl group), as a hydrophilic terminus, provides moderate electronegativity (enhancing stability through electrostatic repulsion) and effectively prevents particle aggregation through the steric hindrance effect of the PEG chain. This robust "anchor-extension" structure ensures the integrity of nanoparticle formulations during storage, while also endowing them with excellent imaging properties and good biocompatibility.
[0039] Research Summary: Based on a systematic comparison of experimental data, DP-PEG-COOH was established as the optimal surface modification scheme.
[0040] The specific reasons are as follows: The suspension exhibits excellent stability: the smallest particle size, narrowest particle size distribution, longest suspension stability days, and moderate negative charge (Zeta potential approximately -21.5 mV). Combined with the steric hindrance effect of the long PEG chain, it effectively resists electrolyte compression and serum protein adsorption, endowing the nanosuspension with excellent physical stability, long-term storage stability, and serum stability. This indicates that DP-PEG-COOH can form the most stable nanosuspension system.
[0041] Excellent imaging performance: It exhibits the highest signal intensity in both CT and MRI (T1, T2) imaging, which is crucial for the application of therapeutic nanoparticles.
[0042] Excellent biocompatibility: The robust PEGylated layer reduces non-specific interactions with cells, resulting in a cell survival rate of nearly 80% and optimal biocompatibility.
[0043] Stable molecular structure: The strong anchoring ability of the DP group combined with the stable hydrophilic protection of the COOH group forms an ideal "anchor-protection" molecular architecture, ensuring that the PEG-modified layer adheres firmly to the surface of the nanoparticles and is not easily detached. This is the fundamental reason why its overall performance is significantly superior to other solutions.
[0044] 2. Characterization results of nanosuspensions Physicochemical properties and characterization of MDPBMNs (abbreviation for MB / (DP-PEG)-BSA@MnO2 nanoparticles) The hydration particle size, PDI, and potential of (DP-PEG)-BSA@MnO2 and MDPBMNs were measured using DLS, and the long-term stability of MDPBMNs was verified by visual observation, Tyndall effect, and sedimentation-resuspension experiments. The microstructure and surface structure of MDPBMNs were observed using TEM and AFM. The physicochemical structure and morphology of MDPBMNs were observed using FTIR and XRD, verifying the successful preparation of MDPBMNs. The elemental composition and quantitative analysis of the nanoparticles were performed using XPS.
[0045] like Figure 2 As shown, MDPBMNs are dark blue-brown, (DP-PEG)-BSA@MnO2 is dark brown, and MB injection solution is dark blue.
[0046] like Figure 3 and Figure 4 As shown, the TEM and mapping images visually demonstrate the morphological characteristics. The average particle size of MDPBMNs is approximately 80-90 nm. As shown in Figure 5, AFM analysis revealed that the surface height of most particles varied from approximately 0.0 nm to 4.0 nm, with the lowest local height reaching -1.3 nm. This indicates that the particles possess excellent planarity and uniformity.
[0047] like Figure 6 and Figure 7 As shown, Figure 6 The spectrum matches the characteristic diffraction peaks of standard δ-MnO2, indicating that MnO2 nanonuclei with a well-defined crystal structure have been successfully synthesized. Figure 7 The simultaneous appearance of characteristic absorption peaks for BSA, DP-PEG, and MB in the spectrum indicates that the three components combine with the MnO2 core through chemical interaction to form a composite nanostructure. These two figures confirm the successful preparation of MDPBMNs.
[0048] like Figure 8-11 As shown, XPS analysis revealed that Mn content was the highest, approximately 74.55%, while Cl and S contents were extremely low (<1%). The O element spectrum indicated a lattice oxygen content >70%, confirming that the particle core is high-purity δ-MnO2.
[0049] like Figure 12 and Figure 13 As shown, DLS results indicate that the hydrated particle sizes of the freshly prepared (DP-PEG)-BSA@MnO2 and MDPPBMNs exhibit a normal distribution. Both have relatively narrow particle size ranges, demonstrating high particle uniformity.
[0050] Suspension stability After the 150ug / mL MB / (DP-PEG)-BSA@MnO2 nano-suspension was allowed to stand for 24 hours, no obvious precipitate was observed at the bottom of the solution, and the solution was in a uniform suspension state. After the nano-suspension was allowed to stand for 30 days, a small amount of precipitate was observed at the bottom of the test tube. After shaking it again, the precipitate at the bottom of the solution disappeared, and the particles were suspended again.
[0051] Particle size and potential DLS analysis showed that the hydrated particle size of the final product was 70-100 nm, the PDI was less than 0.2, and the potential was approximately -20 mV, indicating that the system was stable and homogeneous. TEM and AFM showed that the nanoparticles were spherical or near-spherical and uniformly distributed.
[0052] Cytotoxicity test results After L929 cells were treated with (DP-PEG)-BSA@MnO2 and MB / (DP-PEG)-BSA@MnO2 for 24 h, cell viability showed a slight decreasing trend with increasing Mn2+ concentration, but the decrease was less than 20%, and the cell survival rate of each group remained greater than 75%, with no statistically significant difference between the two groups (p>0.05). This suggests that regardless of whether methylene blue is bound, the carrier does not cause significant acute cytotoxicity when Mn2+ ≤150 µg / mL, meeting the basic requirements for the biosafety of contrast agents. The IC50 was higher than the upper limit of the experimental concentration, which may be due to the surface BSA coating shielding the potential oxidative stress of the MnO2 core, while the physically bound MB showed good chemical stability and did not release additional toxic byproducts. This result provides a reliable dose window for subsequent large animal imaging studies.
[0053] Calcein-AM / PI double staining results Green fluorescence occupied >80% of the field of view, while red fluorescence of PI was sparse, indicating that the nanosuspension caused minimal damage to the cell membrane barrier within 24 hours, and cells maintained normal metabolism. This is consistent with the previous MTT result of >75% cell viability. After 48 hours, the intensity of green fluorescence decreased slightly, and isolated punctate red nuclear staining appeared locally. The proportion of dead cells in the control group and the five groups with Mn2+ concentrations less than 125 µg / mL remained <20%. Only the group with an Mn2+ concentration of 150 µg / mL had a dead cell proportion greater than 20% (approximately 23.57%), but this was still less than the safety limit of 25%. In summary, from a cytological perspective, the MB / (DP-PEG)-BSA@MnO2 nanosuspension exhibits low toxicity, and cell membrane integrity and esterase activity remain largely intact, meeting the biosafety requirements for long-term in vivo imaging.
[0054] Results of in vivo toxicity experiments in SD rats All mice survived normally and maintained good daily activities. The body weight of mice injected with MB / (DP-PEG)-BSA@MnO2 nanosuspension at concentrations of 100 μg / mL and 125 μg / mL increased with the duration of the experiment. The body weight of the 150 μg / mL group did not change significantly within 15 days, but decreased slightly by approximately 1.28% at 30 days, which may be due to objective factors such as feeding or food intake.
[0055] There was no statistically significant difference in organ coefficients between the injection concentration of 150 ug / mL MB / (DP-PEG)-BSA@MnO2 nanosuspension and the control group (P > 0.05). H&E staining results of major organs showed no significant pathological changes at the injection site or in the major organs after injection of 150 ug / mL MB / (DP-PEG)-BSA@MnO2 nanosuspension.
[0056] Within 30 days after injection of the nanosuspension, the levels of MDA, SOD, and CAT in all five major organs showed no significant changes, and there was no significant difference compared with the blank control group (P>0.05). This indicates that the nanosuspension did not induce significant oxidative stress under the experimental conditions, demonstrating good biocompatibility. Furthermore, the oxidative stress level induced by the MB / (DP-PEG)-BSA@MnO2 nanosuspension was not statistically different from that of two commonly used clinical imaging contrast agents (P>0.05).
[0057] GSH Experimental Results Previous studies have shown that the gastrointestinal tract in vivo is rich in GSH, providing sufficient theoretical support for the injection imaging of this suspension into the gastrointestinal serosa. While keeping the GSH level and the concentration of MB / (DP-PEG)-BSA@MnO2 nanosuspension (150 µg / mL) constant, we found that within the pH range of 5-7, the release ratio of Mn2+ gradually increased with decreasing pH. At pH 5, Mn2+ release... 2+The release rates were higher than those of other pH groups at the same time point, reaching a peak of approximately 45.69% at 120 minutes. At pH 7, the average release rate dropped to its lowest point, about half the peak (21.39%). The effect of different GSH concentrations on Mn2+ release was investigated while maintaining pH 6.5 and a suspension concentration of 150 µg / mL. The results showed that the release rate of Mn2+ increased significantly with increasing GSH concentration. Without GSH, the average Mn2+ release rate at 120 minutes was only 35.19%, while under 4 mg GSH conditions, the average release rate increased to 98.94%. This indicates that GSH, as a direct reducing agent, can effectively promote the reduction of MnO2, thereby increasing the release of Mn2+. The higher the GSH concentration, the stronger its reducing power, and the greater the release of Mn2+. 2+ The more MnO2 is released, the better. Keeping pH 6.5 and 4 mM GSH constant, we investigated the effects of different concentration gradients of MB / (DP-PEG)-BSA@MnO2 nanosuspensions on MnO2. 2+ The effects of release. Results showed that Mn 2+ The increase in concentration had little effect on the Mn2+ release ratio. Within the concentration range of 25-150 µg / mL, the average Mn2+ release ratio at 120 minutes showed only a slight increase (97.71%-99.29%).
[0058] In vitro imaging results CT results showed that as the Mn2+ concentration increased from 25 µg / mL to 150 µg / mL, MB / (DP-PEG)-BSA... The CT value of the @MnO2 nanosuspension increased from 59.7 HU to 446.57 HU. At a concentration of 150 µg / mL, the CT value of this nanosuspension was 446.57 HU, while the CT value of iohexol was only 76.63 HU. Compared with iohexol injection, a commonly used CT contrast agent in clinical practice, this nanosuspension showed a higher CT value at the same metal concentration, and overall exhibited superior contrast and CT imaging enhancement capabilities.
[0059] At 3.0 TMR, the MB / (DP-PEG)-BSA@MnO2 nanosuspension also exhibited excellent contrast enhancement performance. With increasing concentrations of both the suspension and Gd-DTPA, the T1 relaxation efficiency (1 / T1) showed a significant increasing trend, while the T2 relaxation efficiency (1 / T2) showed a decreasing trend. Compared to clinically commonly used Gd-DTPA, this nanosuspension at the same concentration demonstrated superior T1 and T2 imaging contrast and sensitivity. At a concentration of 150 µg / mL, 1 / T1 reached 12.3 [Mn] mMs⁻¹ (approximately 3.65 times that of Gd-DTPA at the same concentration), and 1 / T2 reached 19.5 [Mn] mMs⁻¹ (approximately 4.63 times that of Gd-DTPA at the same concentration).
[0060] Results of ex vivo organ injection CT results showed that the MB / (DP-PEG)-BSA@MnO2 nanosuspension exhibited a significant dose-response effect. The nanosuspension at a concentration of 150 µg / mL showed the best imaging (approximately 448.2 HU), while the CT values for 125 µg / mL and 100 µg / mL nanosuspensions were 377.2 HU and 262.8 HU, respectively. The 150 µg / mL iohexol injection had a CT value of 79.2 HU, slightly weaker than the three concentrations of nanosuspension, but still showed significant contrast with surrounding tissues. Gd-DTPA did not show significant imaging in the colon specimen.
[0061] MR imaging on T1 sequences showed that the 150 µg / mL nanosuspension exhibited the most significant imaging, with the highest 1 / T1 ratio (11.88 [Mn] mMs⁻¹), approximately 3.4 times higher than the 1 / T1 ratio of Gd-DTPA at the same concentration (3.49 [Mn] mMs⁻¹). The 1 / T1 ratios of the 125 µg / mL and 100 µg / mL nanosuspensions were slightly lower, at 7.88 [Mn] mMs⁻¹ and 9.75 [Mn] mMs⁻¹, respectively, but both showed significant imaging on ex vivo imaging. No significant imaging was observed on MR with the 150 µg / mL iohexol injection. On T2 sequences, no significant imaging was observed with any concentration of MB / (DP-PEG)-BSA@MnO₂ nanosuspension, iohexol injection, or Gd-DTPA.
[0062] Macroscopic observation revealed that 24 hours after injection of different concentrations of MB / (DP-PEG)-BSA@MnO2 nanosuspension, no large-area staining was observed at any injection site. Results showed that the staining in the 100µg / mL group increased significantly after 12 hours of injection, with a statistically significant difference (1.10±0.19cm vs. 1.68±0.21cm, P<0.05). There were no statistically significant differences between the 125µg / mL and 150µg / mL groups before and after injection (150µg / mL: 1.02±0.15cm vs. 1.27±0.13cm, P>0.05; 125µg / mL: 1.15±0.11cm vs. 1.31±0.14cm, P>0.05). Furthermore, because iohexol injection and Gd-DTPA are colorless and transparent liquids, the size of the staining could not be directly distinguished by the naked eye. Therefore, we roughly determined the size of the halo by examining the imaging size. The results showed that on CT, the width of the halo in the iohexol group was 1.71±0.18 cm, significantly larger than that of the nano-suspension group. MR imaging showed that the halo size in the Gd-DTPA group was 1.98±0.22 cm, similar to the 100 and 125 µg / mL nano-suspension groups, but significantly larger than that of the 150 µg / mL suspension group.
[0063] In vivo efficacy results After anesthetizing Bama miniature pigs, a midline incision was made in the upper abdomen, and 0.1 mL of MB / (DP-PEG)-BSA@MnO2 nanosuspension was injected into the stomach, colon, and small intestine, respectively. The injection sites were clearly visible, and no significant diffusion or spillage occurred, proving successful immediate labeling. Overall, at the same dose, the diameter of the diffusion at different sites in the experimental group was smaller than that in the control group, and the difference was statistically significant. In the stomach tissue, the diameter of the halo in the experimental group was (0.77±0.15) cm, while that in the control group was (1.57±0.35) cm, with a statistically significant difference (P<0.05, P=0.0036). In the small intestine, the diameter of the halo in the experimental group was (0.61±0.16) cm, while that in the control group was (1.57±0.23) cm, with a statistically significant difference (P<0.001, P=0.0016). In the colon, the diameter of the halo in the experimental group was (0.59±0.12) cm, while that in the control group was (1.67±0.25) cm, with a similarly statistically significant difference (P<0.01, P=0.0044).
[0064] CT and MRI scans were performed within 30 minutes of injection. We found significant contrast enhancement at each injection site and surrounding area. Specifically, the CT value for the gastric serosa was 451.7 HU, for the small intestinal serosa 439.9 HU, and for the colonic serosa 432.1 HU. Similar contrast enhancement was observed on MRI: the 1 / T1 ratio for the gastric serosa was 12.6 [Mn]mMs⁻¹, for the small intestinal serosa 11.9 HU, and for the colonic serosa 12.1 HU. Subsequent CT and MRI scans were performed on days 3, 7, and 30 post-injection. Results showed clear imaging at all injection sites within 7 days, with the most significant enhancement observed on day 3. However, with increasing injection time, the CT and 1 / T1 values gradually decreased, and the contrast enhancement gradually weakened. (CT and 1 / T1 values of the stomach, small intestine, and colon on CT / MRI are shown.) On day 30, the original injection sites on CT / MRI had all disappeared. An exploratory laparotomy was then performed through the original surgical incision, and no previously injected gastric, small intestinal, or colonic markers were found, indicating that the long-term staining efficacy still needs improvement. The control group showed no imaging activity. After 30 days, the same exploratory laparotomy through the original surgical incision revealed significant black staining at each marker. This demonstrates that while the nano-carbon suspension lacks imaging enhancement and is prone to smudging compared to MB / (DP-PEG)-BSA@MnO2 nano-suspension, it significantly prolongs the staining time.
[0065] Safety results in Bama miniature pigs Overall, all the experimental Bama miniature pigs showed good feeding and activity levels, and their wounds healed well. After injection of the nanosuspension (150 ug / mL), all tested hematological parameters were within the normal range. This indicates that it did not cause significant systemic toxicity at the experimental dose.
[0066] Thirty days after injection of MB / (DP-PEG)-BSA@MnO2 nanosuspension (150ug / mL) into the serous layer, the organ coefficients of all major organs were within the normal range. There was no statistically significant difference between the experimental group and the control group (P>0.05). No obvious inflammatory reactions or pathological changes were observed in the surrounding tissues, and the tissue structure was not significantly different from the control group. Furthermore, no significant pathological changes were observed in the major organs of the body after injection compared to the control group.
[0067] Metabolic pathway results Mn2+ rapidly accumulated in the liver and kidneys within 2 hours after injection, then decreased rapidly over 24 hours and 7 days. Very small amounts accumulated in other major organs (heart, spleen, and lungs), exhibiting a similar negative time-dose effect. By day 7, Mn2+ concentrations in all organs had decreased to levels similar to the control group (no injection), with no statistically significant difference (P > 0.05). These results indicate that Mn2+ in the MB / (DP-PEG)-BSA@MnO2 nanosuspension is primarily metabolized and excreted through the liver and kidneys, and is nearly completely metabolized (>95%) after one week. This suggests that the nanosuspension does not remain in the body long-term and thus does not affect organs.
Claims
1. A method for preparing albumin-based polyethylene glycol diphosphate modified manganese dioxide methylene blue nanosuspension, characterized in that, Includes the following steps: S1: Preparation of BSA@MnO2: KMnO4 solution was slowly added to MnCl2·4H2O solution to form MnO2 precursor solution, and then the pH of MnO2 precursor solution was adjusted to 6; the pH-adjusted MnO2 precursor solution was subjected to ultrafiltration to separate MnO2 precipitate. The MnO2 precipitate was then washed and dried. The treated MnO2 precipitate was dispersed in pure water to prepare an MnO2 solution. The MnO2 solution and BSA solution were mixed at a volume ratio of 1:5 to form a BSA@MnO2 mixed solution. The BSA@MnO2 mixed solution was subjected to water bath ultrasonic treatment to obtain a stable BSA@MnO2 nanoparticle suspension dispersed in the liquid phase. S2: DP-PEG modification treatment: DP-PEG solid powder was dissolved in pure water and subjected to ultrafiltration and centrifugation to obtain DP-PEG solution. Then, LDP-PEG solution was slowly added to the prepared BSA@MnO2 nano suspension and stirred for 4 hours to obtain (DP-PEG)-BSA@MnO2 transition solution. The (DP-PEG)-BSA@MnO2 transition solution was then subjected to ultrafiltration and centrifugation to obtain the (DP-PEG)-BSA@MnO2 solution. S3: Methylene blue loading treatment: Methylene blue powder is dissolved in pure water and subjected to ultrafiltration and centrifugation to obtain an aqueous solution of methylene blue; The pH of the (DP-PEG)-BSA@MnO2 solution was adjusted to 7-8.5 to make its surface negatively charged, and the pH of the methylene blue aqueous solution was adjusted to 7 to make its surface positively charged. A certain volume of methylene blue solution and (DP-PEG)-BSA@MnO2 solution were mixed to ensure that the mass ratio of methylene blue to BSA@MnO2 in the mixed solution was maintained at 1:
3. Finally, the (DP-PEG)-BSA@MnO2 solution and the methylene blue aqueous solution were mixed at a mass ratio of 1:3 and a concentration ratio of 3:1 and stirred continuously for 6 hours to obtain MB / (DP-PEG)-BSA@MnO2 nano-suspension.
2. The preparation method according to claim 1, characterized in that: In step S1, the concentration of the KMnO4 solution is 4 mg / mL, and the concentration of the MnCl2·4H2O solution is 4 mg / mL.
3. The preparation method according to claim 1, characterized in that: In step S1, the concentration of the MnO2 solution is 4 mg / mL and the concentration of the BSA solution is 20 mg / mL.
4. The preparation method according to claim 1, characterized in that: In step S2, the concentration of the DP-PEG solution is 5 mg / mL.
5. The preparation method according to claim 1, characterized in that: The volume of the LDP-PEG solution in step S2 is 160 μL.
6. The preparation method according to claim 1, characterized in that: The ultrafiltration operation in step S2 shall be performed at least twice.
7. The preparation method according to claim 1, characterized in that: In step S2, the centrifugation operation is performed at least three times, with a centrifugation speed of 400 r / min and a duration of 10 min for each centrifugation operation.
8. The preparation method according to claim 1, characterized in that: In step S3, the concentration of the methylene blue aqueous solution is 1 mg / mL.
9. A protein-based poly(ethylene glycol diphosphate) modified manganese dioxide methylene blue nanosuspension composition, characterized in that: It is prepared by the method according to any one of claims 1-8.