Silk fibroin microspheres loaded with doxorubicin hydrochloride and cyclophosphamide and a preparation method thereof

Abstract

The application discloses a silk fibroin microsphere loaded with doxorubicin hydrochloride and cyclophosphamide and a preparation method of the silk fibroin microsphere. The preparation method comprises the following steps: adding doxorubicin hydrochloride into a silk fibroin solution to obtain a SF / DOX composite system, dissolving cyclophosphamide in an organic solvent to obtain an organic phase composite system, mixing the two through a nano precipitation method, and centrifuging and ultrasonicating to obtain DOX / CTX-SFNPs microspheres. The microspheres keep the stable proportion of the two drugs and enhance the synergistic effect of the combined chemotherapy.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a silk fibroin microsphere loaded with doxorubicin hydrochloride and cyclophosphamide, and its preparation method. Background Technology

[0002] Breast cancer (BC) is one of the most common malignant tumors in women, and radiotherapy and chemotherapy remain important treatment methods. Cyclophosphamide (CTX) and doxorubicin hydrochloride (DOX) are commonly used first-line anti-breast cancer drugs in clinical practice, and are often used in combination to enhance the anti-tumor effect. However, under traditional intravenous or conventional administration methods, the drug is distributed non-specifically in the body, with limited accumulation efficiency at the tumor site, and is prone to producing toxic side effects on normal tissues. In particular, DOX has a certain risk of cardiotoxicity and cannot be used for a long time or at high doses.

[0003] In recent years, nanomedicine delivery systems have been used to improve the in vivo distribution and release behavior of chemotherapeutic drugs. Various nanocarriers, such as liposomes, polymer micelles, metal-organic frameworks, and inorganic nanomaterials, have been reported for single or combined delivery of DOX or CTX. While these technologies have prolonged drug release time or achieved stimulus-response release to some extent, they generally suffer from problems such as complex material composition, cumbersome preparation processes, difficulty in batch stability control, and potential biosafety risks, hindering large-scale production and clinical translation. Furthermore, the significant differences in physicochemical properties between different drugs in dual-drug co-loading systems can easily lead to unstable drug loading ratios or asynchronous release, thus affecting the synergistic effect of combined chemotherapy.

[0004] Silk fibroin (SF), an abundant natural polymer derived from silkworm cocoons, possesses excellent biocompatibility, biodegradability, and structural tunability. It can be prepared in various forms such as films, gels, porous scaffolds, microneedles, and particles, and has been applied in tissue engineering and drug delivery. Chinese invention patent CN105597104B describes the preparation of silk fibroin microspheres by blending polyethylene glycol and an aqueous solution of silk fibroin to form an emulsion. This method has a relatively simple process, does not require the introduction of organic solvents, and exhibits good biocompatibility. However, drug-loaded micro / nanospheres prepared using this method suffer from a relatively rapid drug release rate when loaded with small molecule drugs, making precise control of drug release behavior difficult.

[0005] In summary, the existing technology lacks a silk fibroin microsphere system with a relatively simple structure, controllable preparation process, and the ability to stably co-encapsulate DOX and CTX and achieve synergistic sustained release. Summary of the Invention

[0006] Objective: To overcome the shortcomings of existing technologies, this invention provides silk fibroin microspheres loaded with doxorubicin hydrochloride and cyclophosphamide, and a method for preparing the same, which improves the effective delivery and retention of drugs at tumor sites, reduces systemic toxicity, and enhances the therapeutic effect of combined chemotherapy.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for preparing silk fibroin microspheres loaded with doxorubicin hydrochloride and cyclophosphamide, comprising: S1. Add doxorubicin hydrochloride to the silk fibroin solution to obtain the SF / DOX composite system; dissolve cyclophosphamide in an organic solvent to obtain an organic phase composite system; S2. The SF / DOX composite system and the organic phase composite system are mixed by nanoprecipitation to obtain a silk fibroin nanoparticle suspension loaded with doxorubicin hydrochloride and cyclophosphamide. S3. Centrifugation, sonication, and drying yield DOX / CTX-SFNPs microspheres.

[0009] Silk fibroin microspheres loaded with doxorubicin hydrochloride and cyclophosphamide comprise multiple drug-loaded nanoparticles and multiple silk fibroin microparticles, with the drug-loaded nanoparticles dispersed within the silk fibroin microparticles. The silk fibroin microparticles are formed by multiple silk fibroin molecules I; the drug-loaded nanoparticles comprise doxorubicin hydrochloride, cyclophosphamide, and multiple silk fibroin molecules II. Specifically, silk fibroin molecules I and II have identical structures, serving only to distinguish between the silk fibroin molecules forming the silk fibroin microparticles and those forming the drug-loaded nanoparticles.

[0010] During the preparation process, DOX and SF first form an SF / DOX composite system in the aqueous phase. DOX binds to SF molecules through electrostatic interactions (DOX, being positively charged, interacts with carboxyl / anionic groups on SF molecules), hydrogen bonding, and hydrophobic interactions, and is dispersed inside SF nanoparticles during the subsequent nanoprecipitation process. CTX is pre-dissolved in solvents such as acetone to form an organic phase composite system. During the mixing and self-assembly of the aqueous and organic phases, CTX diffuses into the forming SF nanoparticles along with the solvent, and finally co-encapsulates with DOX.

[0011] In the resulting microspheres, doxorubicin hydrochloride, cyclophosphamide, and silk fibroin are distributed in one or a combination of the following forms: (1) Doxorubicin hydrochloride and cyclophosphamide are dispersed inside silk fibroin nanoparticles formed by multiple silk fibroin molecules; (2) Doxorubicin hydrochloride is relatively enriched in the interior or near-surface region of silk fibroin nanoparticles, while cyclophosphamide is relatively enriched in the hydrophobic region inside the silk fibroin nanoparticles formed by multiple silk fibroin molecules. (3) A portion of doxorubicin hydrochloride and / or cyclophosphamide are encapsulated inside the silk fibroin nanoparticles, while the other portion is distributed on the surface or near the surface of the silk fibroin nanoparticles by adsorption and / or weak binding.

[0012] Silk fibroin nanoparticles are formed by the conformational transformation and self-assembly of silk fibroin molecules induced by desolventization. Preferably, they consist of a network structure containing both β-sheet crystalline regions and amorphous regions. The β-sheet crystalline regions serve as physical cross-linking points, enhancing particle density and structural stability, while the amorphous regions provide diffusion channels for drugs and participate in subsequent degradation and drug release processes. The degradation process of DOX / CTX-SFNPs exhibits a typical two-stage characteristic: an initial rapid release phase and a later sustained-release equilibrium phase.

[0013] The preferred β-sheet content is increased by 10%-20%; the relative content of the random coil structure is reduced accordingly, thereby improving the density of the nanoparticle structure and delaying the drug diffusion rate.

[0014] In some embodiments, the silk fibroin solution contains 0.5-1 wt% silk fibroin, preferably 0.75 wt%.

[0015] In some embodiments, the mass ratio of doxorubicin hydrochloride and cyclophosphamide in S1 is 1:10-5, preferably 1:5.

[0016] In some embodiments, the preparation method of the organic phase composite system in S1 includes: dissolving cyclophosphamide in an organic solvent and stirring for 5-60 min; and storing the organic phase composite system at 2-6°C in the dark.

[0017] The organic solvent is selected from acetone, ethylene glycol, and ethanol.

[0018] In some embodiments, the mixing of the SF / DOX composite system and the organic phase composite system by nanoprecipitation in S2 includes: mixing the SF / DOX composite system and the organic phase composite system at a volume ratio of 1:5-20, and stirring and incubating at 10-35°C at a constant speed of 500-800 rpm for 10-30 min.

[0019] In some embodiments, the centrifugation conditions in S3 include: centrifugation at 8000-20000 rpm for 3-10 min; and the centrifugation temperature is 0-20℃.

[0020] Preferably, the centrifugation speed is 10000-12000 rpm, the duration is 3-5 min, and the temperature is 4℃.

[0021] More preferably, the centrifugation speed is 12,000 rpm and the duration is 5 min.

[0022] In some embodiments, the power of the ultrasound in S3 is 100-200 W, and the duration is 1-5 min.

[0023] In some embodiments, the number of ultrasound sessions in S3 is 2-6, preferably 3.

[0024] In some embodiments, the ultrasonic treatment in S3 further includes a drying step; the drying is freeze-drying, and the freeze-drying method includes: pre-freezing at -80°C for 24-48 h, followed by freeze-drying for 36-60 h.

[0025] In a second aspect, the present invention provides silk fibroin microspheres loaded with doxorubicin hydrochloride and cyclophosphamide, which are prepared by the preparation method described in the first aspect.

[0026] Silk fibroin microspheres loaded with doxorubicin hydrochloride and cyclophosphamide, prepared using the method described in the first aspect, maintained an encapsulation efficiency of over 80% for CTX, with no statistically significant differences between groups; the encapsulation efficiency of DOX in the aqueous system ranged from 55% to 95%. The total sustained-release time of the microspheres in the in vitro release experiment was ≥100 h; within the initial 12 h, the maximum weight loss rate of the sample reached 44.75% ± 4.52%; subsequently, in the range of 24 h to 96 h, the degradation rate showed a slowing trend, and the final cumulative weight loss rate stabilized at 86.90% ± 2.31%.

[0027] In some embodiments, the silk fibroin microspheres loaded with doxorubicin hydrochloride and cyclophosphamide have a particle size of 70-200 nm.

[0028] The microspheres have a spherical or near-spherical structure, are uniformly dispersed, and show no obvious aggregation.

[0029] Beneficial Effects: The DOX / CTX-SFNPs provided by this invention use silk fibroin as a single carrier material and achieve stable co-encapsulation of DOX and CTX through a "phase-separated stepwise loading-nanoprecipitation self-assembly" method, enabling the simultaneous delivery of two small molecule drugs with different physicochemical properties in the same nanoparticle. This structural design helps maintain a stable ratio of the two drugs, reduces ratio fluctuations and release asynchrony problems caused by simple blending, thereby enhancing the synergistic effect of combined chemotherapy and reducing systemic toxic side effects caused by free drugs to a certain extent.

[0030] This invention induces the transformation of silk fibroin from random coils to β-sheet structures by controlling the concentration, solvent ratio, and self-assembly conditions, forming a relatively dense nanomatrix structure. This effectively slows down the drug diffusion rate and achieves controlled sustained release. The method is relatively simple, with adjustable parameters, and exhibits good reproducibility and scalability potential. Attached Figure Description

[0031] Figure 1 This is a schematic diagram illustrating the formation process of DOX / CTX-SFNPs in an embodiment of the present invention.

[0032] Figure 2 The figures show the surface morphology of DOX / CTX-SFNPs in the embodiments of the present invention; wherein, ai in the figures are the surface morphology of DOX / CTX-SFNPs prepared in Examples 1-9, respectively.

[0033] Figure 3 The figures show the yield analysis of different silk fibroin microspheres prepared in the embodiments and comparative examples of the present invention; wherein, groups E1-E3 are drug-free silk fibroin nanospheres prepared in Comparative Example 1 (silk fibroin solution mass fractions of 0.5 wt.%, 0.75 wt.%, and 1 wt.%); groups 1-9 are DOX / CTX-SFNPs prepared in Examples 1-9.

[0034] Figure 4 This is a graph showing the average particle size analysis of different silk fibroin microspheres in the embodiments and comparative examples of the present invention. Figure 4 a) PDI analysis chart ( Figure 4 (b) and Zeta analysis plot ( Figure 4 (c) Among them, E1-E3 are drug-free silk fibroin nanospheres prepared in Comparative Example 1 (silk fibroin solution mass fraction of 0.5 wt.%, 0.75 wt.% and 1 wt.%); 1-9 are DOX / CTX-SFNPs prepared in Examples 1-9.

[0035] Figure 5 The graph shows the weight loss of DOX / CTX-SFNPs prepared in the embodiments of the present invention over time in PBS and simulated gastric juice (SGF).

[0036] Figure 6 This is a graph showing the encapsulation efficiency and drug loading rate test results of DOX or CTX in embodiments of the present invention; wherein, Figure 6 Figures a and b show the encapsulation efficiency and drug loading rate of DOX in the DOX / CTX-SFNPs prepared in Examples 1-9. Figure 6 Figures c and d show the encapsulation efficiency and drug loading rate of CTX in DOX / CTX-SFNPs prepared in Examples 1-9.

[0037] Figure 7 These are photographs of tumor-bearing mice in different treatment groups in this invention.

[0038] Figure 8 This is a flowchart of mouse processing in an embodiment of the present invention. Figure 8 (a) and statistical graph of mouse body weight change ( Figure 8 (b)

[0039] Figure 9 These are tumor photographs of tumor-bearing mice in different treatment groups in this invention embodiment. Figure 9 (a) and statistical graph of changes in mouse tumor volume ( Figure 9 (b) Detailed Implementation

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use.

[0041] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may include different values. It should be noted that similar reference numerals and letters in the following figures denote similar items; therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0042] The present invention will be further described below with reference to the embodiments.

[0043] In the following examples, all reagents and materials used were readily available; all quantitative experiments were performed at least three times, and the results were averaged.

[0044] Example 1

[0045] This embodiment provides a method for preparing silk fibroin microspheres (DOX / CTX-SFNPs) loaded with doxorubicin hydrochloride and cyclophosphamide. The formation process of DOX / CTX-SFNPs is as follows: Figure 1 As shown, the method includes:

[0046] (1) Preparation of silk fibroin solution. 4 L of deionized water was heated to boiling, and 8.48 g of sodium carbonate was added and stirred until completely dissolved. Then, cleaned silkworm cocoons (approximately 10 g in weight) were cut into small pieces and immersed in boiling water for 30 minutes to degumme. The degummed silk was washed and air-dried overnight. The air-dried silk was added to LiBr solution and dissolved at 60°C for 4 hours to obtain a homogeneous silk fibroin solution. The obtained solution was dialyzed for 36 hours and filtered to remove LiBr and other small molecule salts, and further filtered to remove insoluble impurities, obtaining a molecularly intact and homogeneous silk fibroin solution, providing a basis for the self-assembly of nanoparticles.

[0047] (2) Preparation of lyophilized silk fibroin powder. The silk fibroin solution obtained in step (1) was prepared into a 3 wt% silk fibroin solution, and aliquoted with 0.1 M PBS buffer (pH=7.4) at a volume ratio of 20:1 into transparent vials, 10 mL per vial. After sealing, the vials were placed on a liquid nitrogen pre-freezing plate for rapid freezing. After lyophilization for 96 hours, dry silk fibroin powder was obtained. This lyophilized powder can be stored for a long time while maintaining the integrity of the protein molecular structure, which facilitates the subsequent self-assembly of nanoparticles.

[0048] (3) Preparation of drug-loaded silk fibroin microspheres. The lyophilized powder obtained in step (2) was reconstituted with deionized water to form a 0.5 wt.% silk fibroin solution. DOX was added to the silk fibroin solution to form an SF / DOX composite system with a DOX to CTX mass ratio of 1:5 and a DOX / CTX:SF concentration ratio of 0.1 mg / mL. At the same time, CTX was dissolved in acetone to obtain an acetone / CTX solution. The SF / DOX solution and the acetone / CTX solution were mixed at a volume ratio of 1:9 and stirred at room temperature at a constant speed (700 rpm) for 30 minutes to allow the silk fibroin to self-assemble into drug-loaded nanoparticles, effectively encapsulating DOX and CTX. The resulting precipitate was separated by ultracentrifugation (12000 rpm, 5 minutes), and dispersed by ultrasonication (100 W, 3 minutes) with deionized water and washed three times to remove residual solvent. Pre-freeze at -80℃ for 24 hours, then freeze-dry for 48 hours to obtain dry DOX / CTX-SFNPs powder, and store it in a sealed container at 4℃ in the dark.

[0049] The resulting DOX / CTX-SFNPs have a particle size of 50~200 nm. The drug-loaded nanoparticles are dispersed in silk fibroin microspheres. The interaction between silk fibroin molecules forms a stable micron-sized particle structure. The nanoparticles effectively encapsulate DOX and CTX, achieving sustained-release function.

[0050] SEM results of DOX / CTX-SFNPs prepared in this embodiment ( Figure 2As shown in a), the nanoparticles are approximately spherical in shape and have a relatively uniform particle size distribution; Figure 4 As shown, the average particle size is approximately 121.06 nm, the PDI is approximately 0.31, and the Zeta potential is approximately -9.8 mV. FT-IR analysis revealed that silk fibroin underwent a conformational transition from helical and other non-β-sheet forms to β-sheet forms, with a decrease in random coil content and an increase in β-sheet content. Characteristic peaks of DOX and CTX were detected, indicating successful drug encapsulation.

[0051] The prepared DOX / CTX-SFNPs were subjected to in vitro degradation analysis to compare the residual mass of DOX / CTX-SFNPs in PBS and simulated gastric fluid. In this embodiment of the invention, the in vitro degradation analysis method included: weighing 100 mg of DOX / CTX-SFNPs and dissolving them in PBS and simulated gastric fluid respectively to obtain suspensions; aliquoting the suspensions into centrifuge tubes, with each centrifuge tube containing 1 mL of suspension; recording the initial mass W0 (mg) of the empty centrifuge tube and the final mass W1 (mg) of the centrifuge tube containing the suspension; each experiment included three parallel samples, and these centrifuge tubes were sealed and placed on a shaker to maintain a constant temperature of 37°C.

[0052] The method for preparing simulated gastric juice includes: weighing 1.6 g of pepsin and 1.0 g of sodium chloride, dissolving them in deionized water, adjusting the pH to 1.2 with hydrochloric acid, and finally bringing the volume to 500 mL.

[0053] Centrifuge tubes were removed from the shaker at preset time points (20 h, 40 h, 60 h, 80 h, 100 h), centrifuged, and then dried in a 60℃ oven until their mass no longer changed. At this point, the mass of the centrifuge tubes was weighed again and recorded as W2 (mg). The formula for calculating the sample weight residue rate R (%) is: R (%) = (W2 - W0) / (W1 - W2) × 100%.

[0054] The drug loading performance of the prepared DOX / CTX-SFNPs was tested. In this embodiment of the invention, the test method includes:

[0055] (1) Testing the DOX drug loading performance of DOX / CTX-SFNPs:

[0056] Establishing a DOX absorbance-concentration standard curve: DOX was dissolved in distilled water and serially diluted. Using the absorbance of distilled water at 480 nm as the background value, the test values ​​were subtracted from the background value. A scatter plot was then used to plot a fitted line, and a linear regression equation was calculated (with the concentration of the DOX standard solution as the x-axis and the corrected absorbance as the y-axis, concentration points were plotted using the scatter plot method, and the scatter plots were fitted using a linear fitting method to obtain the linear regression equation Y = kX + b, where Y is the corrected absorbance, X is the DOX concentration, k is the slope, and b is the intercept), i.e., the DOX absorbance-concentration standard curve. In this embodiment of the invention, the DOX absorbance-concentration standard curve in aqueous medium is Y = 0.60566X + 0.00302 (R0). 2 =0.9997).

[0057] Weigh 5 mg of DOX / CTX-SFNPs and dissolve them in 5 mL of distilled water. Shake at 37°C for 30 min to ensure complete dissolution. After centrifugation, collect the supernatant and measure the absorbance at 480 nm. Calculate the DOX content using the DOX absorbance-concentration standard curve. The DOX content is denoted as W3.

[0058] The formula for calculating drug loading rate (DLC) is: DLC (%) = W3 / W4 × 100%.

[0059] The formula for calculating the package yield (EE) is: EE (%) = W3 / W5 × 100%.

[0060] In the formula: W3 is the mass of DOX in the sample (mg); W4 is the mass of the sample (mg), in this embodiment, W4=5 mg; W5 is the amount of DOX added (mg).

[0061] (2) Testing the CTX drug loading performance of DOX / CTX-SFNPs:

[0062] Establishing a CTX absorbance-concentration standard curve: CTX was dissolved in ethanol and serially diluted. Using ethanol as the background value, the test value was subtracted from the background value, and the absorbance was measured at 195 nm using a UV spectrophotometer. A fitted line was plotted using the scatter method, and the linear regression equation was calculated (with the concentration of the CTX standard solution as the x-axis and the corrected absorbance as the y-axis, each concentration point was plotted using the scatter method, and the scatter points were fitted using a linear fitting method to obtain the linear regression equation Y=kX+b, where Y is the corrected absorbance, X is the CTX concentration, k is the slope, and b is the intercept), which is the CTX absorbance-concentration standard curve. In this embodiment of the invention, the CTX absorbance-concentration standard curve in ethanol medium is Y=0.34003X+0.00124 (R 2 =0.9999).

[0063] Weigh 5 mg of DOX / CTX-SFNPs and dissolve them in 5 mL of ethanol. Shake at 37°C for 30 min to ensure complete dissolution. After centrifugation, take the supernatant and measure the absorbance at 195 nm. Calculate the CTX content using the CTX absorbance-concentration standard curve. The CTX content is recorded as W6.

[0064] The formula for calculating drug loading rate (DLC) is: DLC (%) = W6 / W7 × 100%.

[0065] The formula for calculating the package yield (EE) is: EE (%) = W6 / W8 × 100%.

[0066] In the formula: W6 is the mass of CTX in the sample (mg); W7 is the mass of the sample (mg), in this embodiment, W7=5 mg; W8 is the amount of CTX added (mg).

[0067] In vitro degradation experiments showed that the residual mass after 24 h in PBS medium was approximately 58%, with rapid initial release and stable sustained release in the later stages. Figure 5 Drug loading performance analysis showed that DOX encapsulation efficiency was approximately 55%-60%, while CTX encapsulation efficiency was above 80%. Drug loading increased with increasing SF concentration and microsphere particle size. Figure 6 ).

[0068] Example 2

[0069] This embodiment provides a method for preparing silk fibroin microspheres (DOX / CTX-SFNPs) loaded with doxorubicin hydrochloride and cyclophosphamide. The difference from Example 1 is that the mass fraction of the silk fibroin solution is 0.5 wt%, the mass ratio of DOX to CTX is 1:7.5, and the concentration ratio of DOX / CTX:SF is 0.2 mg / mL.

[0070] SEM results of DOX / CTX-SFNPs prepared in this embodiment ( Figure 2 As shown in b), the microspheres are all nearly spherical; for example... Figure 4 As shown, the particle size is approximately 106.81 nm, the PDI is approximately 0.39, and the Zeta potential is approximately -7.3 mV, indicating uniform particle dispersion. FT-IR analysis showed that SF underwent a conformational transition from helical and other non-β-sheet configurations to a β-sheet configuration, with reduced random coiling and increased β-sheet. Characteristic peaks for both DOX and CTX were detected, indicating successful drug encapsulation. In vitro degradation experiments showed that the residual mass in PBS medium after 24 hours was approximately 59% (…). Figure 5 ).like Figure 6 As shown, the DOX packaging efficiency is approximately 62%, and the CTX packaging efficiency is approximately 82%, both exhibiting good sustained-release performance.

[0071] Example 3

[0072] This embodiment provides a method for preparing silk fibroin microspheres (DOX / CTX-SFNPs) loaded with doxorubicin hydrochloride and cyclophosphamide. The difference from Example 1 is that the silk fibroin solution has a mass fraction of 0.5 wt%, the DOX:CTX mass ratio is 1:10, and the DOX / CTX:SF concentration ratio is 0.3 mg / mL.

[0073] SEM results of DOX / CTX-SFNPs prepared in this embodiment ( Figure 2 As shown in c), the microspheres are regularly spherical with slight local aggregation; such as Figure 4 As shown, the average particle size is approximately 122.78 nm, the PDI is approximately 0.50, and the Zeta potential is approximately -6.7 mV. FT-IR analysis revealed an increased β-sheet conformation content and a decreased random coil content, indicating effective encapsulation of both DOX and CTX. In vitro degradation experiments showed that approximately 60% of the residual mass remained in the PBS medium after 24 hours. Figure 5 ).like Figure 6 As shown, the DOX packaging efficiency is approximately 68%, and the CTX packaging efficiency is >80%, with uniform and stable particle size.

[0074] Example 4

[0075] This embodiment provides a method for preparing silk fibroin microspheres (DOX / CTX-SFNPs) loaded with doxorubicin hydrochloride and cyclophosphamide. The difference from Example 1 is that the silk fibroin solution has a mass fraction of 0.75 wt%, the DOX:CTX mass ratio is 1:5, and the DOX / CTX:SF concentration ratio is 0.2 mg / mL.

[0076] SEM results of DOX / CTX-SFNPs prepared in this embodiment ( Figure 2 As shown in d), silk fibroin forms spherical particles through self-assembly; such as Figure 4 As shown, the average particle size is approximately 79.81 nm, the PDI is approximately 0.24, and the Zeta potential is approximately -18 mV. FT-IR analysis reveals an increased β-sheet conformation content, indicating successful encapsulation of DOX and CTX. Figure 5 As shown, the residual mass in the PBS medium during the in vitro degradation experiment was approximately 60% after 24 hours. Figure 6 As shown, DOX packaging efficiency is approximately 84%, and CTX packaging efficiency is >80%, with stable particles suitable for continuous release.

[0077] Example 5

[0078] This embodiment provides a method for preparing silk fibroin microspheres (DOX / CTX-SFNPs) loaded with doxorubicin hydrochloride and cyclophosphamide, which differs from Example 1 in that: the silk fibroin solution has a mass fraction of 0.75 wt%, the DOX:CTX mass ratio is 1:7.5, and the DOX / CTX:SF concentration ratio is 0.3 mg / mL. The SEM results of the DOX / CTX-SFNPs prepared in this embodiment are shown below. Figure 2 As shown in (e), the microspheres are round and uniformly distributed; Figure 4 As shown, the particle size is approximately 78.82 nm, the PDI is approximately 0.29, and the Zeta potential is approximately -14 mV. In vitro degradation showed that approximately 60% of the residual mass remained in the PBS medium. Figure 5 As shown in Figure 6, the DOX packaging efficiency is approximately 87%, and the CTX packaging efficiency is >80%, demonstrating good drug loading and sustained-release performance.

[0079] Example 6

[0080] This embodiment provides a method for preparing silk fibroin microspheres (DOX / CTX-SFNPs) loaded with doxorubicin hydrochloride and cyclophosphamide, which differs from Example 1 in that: the silk fibroin solution has a mass fraction of 0.75 wt%, the DOX:CTX mass ratio is 1:10, and the DOX / CTX:SF concentration ratio is 0.1 mg / mL. The SEM results of the DOX / CTX-SFNPs prepared in this embodiment are shown below. Figure 2 As shown in f), the microspheres are nearly spherical; as Figure 4 As shown, the particle size is approximately 77.74 nm, the PDI is approximately 0.37, and the Zeta potential is approximately -17.5 mV. Figure 5 As shown, the residual mass of the PBS medium after 24 hours of in vitro degradation is approximately 60%. Figure 6 As shown, the DOX packaging efficiency is approximately 85%, and the CTX packaging efficiency is >80%, with uniform and stable particle size.

[0081] Example 7

[0082] This embodiment provides a method for preparing silk fibroin microspheres (DOX / CTX-SFNPs) loaded with doxorubicin hydrochloride and cyclophosphamide. The difference from Example 1 is that the silk fibroin solution has a mass fraction of 1 wt%, the DOX:CTX mass ratio is 1:5, and the DOX / CTX:SF concentration ratio is 0.3 mg / mL.

[0083] SEM results of DOX / CTX-SFNPs prepared in this embodiment ( Figure 2 (g) shows that silk fibroin self-assembles into a spherical structure; such as Figure 4As shown, the particle size is approximately 120.42 nm, the PDI is approximately 0.15, and the Zeta potential is approximately -26.5 mV. FT-IR showed an increased β-sheet content, indicating that both DOX and CTX were encapsulated. In vitro degradation experiments showed that approximately 97% of the residual mass remained in the PBS medium after 24 hours. Figure 5 ).like Figure 6 As shown, DOX packaging efficiency is approximately 75%, CTX packaging efficiency is >80%, and the chips are stable.

[0084] Example 8

[0085] This embodiment provides a method for preparing silk fibroin microspheres (DOX / CTX-SFNPs) loaded with doxorubicin hydrochloride and cyclophosphamide. The difference from Example 1 is that the silk fibroin solution has a mass fraction of 1 wt%, the DOX:CTX mass ratio is 1:7.5, and the DOX / CTX:SF concentration ratio is 0.1 mg / mL.

[0086] SEM results of DOX / CTX-SFNPs prepared in this embodiment ( Figure 2 As shown in h), the microspheres are uniformly spherical; as Figure 4 As shown, the particle size is approximately 115.02 nm, the PDI is approximately 0.20, and the Zeta potential is approximately -22.5 mV. Figure 5 As shown, the residual mass in the PBS medium during the in vitro degradation experiment was approximately 75% after 24 hours. Figure 6 As shown, DOX has a packaging efficiency of approximately 96%, while CTX has a packaging efficiency of >80%, demonstrating good drug loading performance.

[0087] Example 9

[0088] This embodiment provides a method for preparing silk fibroin microspheres (DOX / CTX-SFNPs) loaded with doxorubicin hydrochloride and cyclophosphamide. The difference from Example 1 is that the silk fibroin solution has a mass fraction of 1 wt%, the DOX:CTX mass ratio is 1:10, and the DOX / CTX:SF concentration ratio is 0.2 mg / mL.

[0089] SEM results of DOX / CTX-SFNPs prepared in this embodiment ( Figure 2 As shown in i), the microspheres are uniformly spherical; as Figure 4 As shown, the particle size is approximately 141.69 nm, the PDI is approximately 0.26, and the Zeta potential is approximately -19.5 mV. Figure 5 As shown, the residual mass of the PBS medium after 24 hours of in vitro degradation is approximately 76%. Figure 6 As shown, DOX has a packaging efficiency of approximately 98%, while CTX has a packaging efficiency of >80%, and the particles are stable and suitable for sustained drug release.

[0090] Example 10

[0091] This embodiment provides a method for preparing silk fibroin microspheres (DOX / CTX-SFNPs) loaded with doxorubicin hydrochloride and cyclophosphamide. The difference from Example 1 is that CTX is dissolved or dispersed in polyethylene glycol to obtain a PEG / CTX solution, and the SF / DOX solution and the PEG / CTX solution are mixed at a volume ratio of 1:9 and stirred at a constant speed for 30 minutes at room temperature to obtain DOX / CTX-SFNPs.

[0092] Example 11

[0093] This embodiment provides a method for preparing silk fibroin microspheres (DOX / CTX-SFNPs) loaded with doxorubicin hydrochloride and cyclophosphamide. The difference from Example 1 is that: CTX is dissolved or dispersed in ethanol to obtain a PEG / CTX solution, and the SF / DOX solution and the PEG / CTX solution are mixed at a volume ratio of 1:9 and stirred at a constant speed for 30 minutes at room temperature to obtain DOX / CTX-SFNPs.

[0094] Comparative Example 1

[0095] This comparative example provides a method for preparing silk fibroin nanospheres without loading doxorubicin hydrochloride and cyclophosphamide.

[0096] Dry silk fibroin powder obtained through freeze-drying was used to prepare silk fibroin solutions with mass fractions of 0.5 wt.%, 0.75 wt.%, and 1 wt.%, respectively, using deionized water. The silk fibroin solution was added dropwise to the pre-prepared acetone at a volume ratio of 1:10, and stirring was continued until a precipitate formed. Subsequently, the mixture was stirred at 700 rpm for 30 min at room temperature to obtain a mixed dispersion containing silk fibroin nanospheres.

[0097] The resulting mixed dispersion was centrifuged at 12,000 rpm for 5 min to separate the generated silk fibroin nanospheres from the dispersion medium. After centrifugation, the supernatant was discarded, and an appropriate amount of deionized water was added to the precipitate. The precipitate was then ultrasonically vibrated to re-disperse it uniformly. Subsequently, the precipitate was centrifuged and washed under the same conditions as in Example 1, and the washing was repeated three times to remove residual acetone. Finally, the washed silk fibroin nanosphere dispersion was pre-frozen at -80°C for 24 h and then freeze-dried for 48 h to obtain three types of silk fibroin nanospheres, labeled E1, E2, and E3. The obtained microspheres were sealed and stored at 4°C in the dark for subsequent testing.

[0098] The yield of silk fibroin microspheres prepared in Examples 1-9 and Comparative Example 1 was analyzed, such as... Figure 3As shown, the yield of drug-free silk fibroin microspheres (E1-E3) increased with increasing silk fibroin concentration. The drug-loaded silk fibroin microspheres prepared in Examples 1-3 had lower yields due to insufficient silk fibroin molecules to adequately encapsulate the drug, resulting in some drug loss during preparation. Increasing the silk fibroin concentration significantly increased the yield of drug-loaded silk fibroin microspheres prepared in Examples 4-9, indicating that silk fibroin concentration is a key factor affecting the efficiency of silk fibroin microsphere formation.

[0099] Example 12

[0100] To evaluate the antitumor effect of DOX / CTX-SFNPs in vivo, a mouse model of MDA-MB-231 triple-negative breast cancer was constructed. After the tumor volume reached the experimental requirements, 16 tumor-bearing mice were randomly divided into four groups (n=4 per group) according to body weight: a saline group, an SFNPs group, a DOX / CTX group, and a DOX / CTX-SFNPs group. Mice in each group were intraperitoneally injected with saline, SFNPs, a DOX / CTX mixture, or DOX / CTX-SFNPs, respectively. The dosage, frequency, and duration of administration followed the pre-designed experimental protocol.

[0101] Animal model drug administration procedure as follows Figure 7 As shown in the figures, the photos and weight changes of the mice after sacrifice in each group are as follows: Figure 8 As shown in the figure. By day 15 of the drug administration observation, differences in body weight were observed among the groups. Specifically, the body weight of mice in the saline group and the SFNPs group showed an increasing trend, increasing from 20.15±0.74 g and 21.26±0.51 g to 25.56±0.61 g and 24.94±0.44 g, respectively; while the body weight of mice in the DOX / CTX group and the DOX / CTX-SFNPs group showed a decreasing trend, decreasing from 21.32±0.56 g and 20.58±0.76 g to 19.12±0.60 g and 19.69±0.15 g, respectively.

[0102] To further evaluate the effects of each treatment group on tumor growth, tumor volume was measured in mice of each group during the drug administration observation period, and tumor growth curves were plotted. After 15 days of drug administration observation, mice were euthanized, and tumor tissue from each treatment group was collected. Figure 9 As shown, the tumor volume in mice in the saline group and the SFNPs group showed an increasing trend, increasing from 33.25 ± 2.43 mm. 3 and 34.56±2.07 mm 3 Increased to 81.53±1.79 mm 3 and 79.42±2.46 mm 3 The tumor volume in the DOX / CTX group mice decreased from 35.62 ± 1.61 mm.3 Reduced to 20.73±2.61 mm 3 The tumor volume in mice in the DOX / CTX-SFNPs group decreased from 35.86 ± 0.12 mm. 3 Reduced to 10.86±4.17 mm 3 The value was the lowest among all treatment groups. These results indicate that DOX / CTX-SFNPs can effectively inhibit tumor growth in MDA-MB-231 tumor-bearing mice.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing silk fibroin microspheres loaded with doxorubicin hydrochloride and cyclophosphamide, characterized in that, include: S1. Add doxorubicin hydrochloride to the silk fibroin solution to obtain the SF / DOX complex system; Cyclophosphamide was dissolved in an organic solvent to obtain an organic phase composite system; S2. The SF / DOX composite system and the organic phase composite system are mixed by nanoprecipitation to obtain a silk fibroin nanoparticle suspension loaded with doxorubicin hydrochloride and cyclophosphamide. S3. Centrifugation and sonication were performed to obtain DOX / CTX-SFNPs microspheres.

2. The preparation method according to claim 1, characterized in that, The silk fibroin solution contains 0.5-1 wt% silk fibroin.

3. The preparation method according to claim 1, characterized in that, The mass ratio of doxorubicin hydrochloride and cyclophosphamide in S1 is 1:10-5.

4. The preparation method according to claim 1, characterized in that, The preparation method of the organic phase composite system described in S1 includes: dissolving cyclophosphamide in an organic solvent and stirring for 5-60 min; storing the organic phase composite system at 2-6℃ in the dark.

5. The preparation method according to claim 1, characterized in that, The mixing of the SF / DOX composite system and the organic phase composite system by nanoprecipitation as described in S2 includes: mixing the SF / DOX composite system and the organic phase composite system at a volume ratio of 1:5-20, and stirring and incubating at a constant speed of 500-800 rpm at 10-35℃ for 10-30 min.

6. The preparation method according to claim 1, characterized in that, The centrifugation conditions described in S3 include: centrifugation at 8000-20000 rpm for 3-10 min; and centrifugation temperature of 0-20℃.

7. The preparation method according to claim 1, characterized in that, The ultrasound power in S3 is 100-200 W, the duration is 1-5 min, and the number of ultrasound sessions is 2-6.

8. The preparation method according to claim 1, characterized in that, The ultrasound step described in S3 also includes a drying step; the drying is freeze-drying, and the freeze-drying method includes: pre-freezing at -80℃ for 24-48 h, and then freeze-drying for 36-60 h.

9. A silk fibroin microsphere loaded with doxorubicin hydrochloride and cyclophosphamide, characterized in that, It is prepared by the preparation method described in any one of claims 1-8.

10. The silk fibroin microspheres loaded with doxorubicin hydrochloride and cyclophosphamide according to claim 9, characterized in that, The silk fibroin microspheres loaded with doxorubicin hydrochloride and cyclophosphamide have a particle size of 70-200 nm.

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