Fh peptide-polydopamine coated graphene oxide drug delivery system and preparation and application thereof

Abstract

This invention relates to the field of biomedical materials and pharmaceutical formulations, disclosing an FH peptide-polydopamine-coated graphene oxide drug delivery system and its preparation and application. The system includes an RGD peptide-modified graphene oxide carrier, a polydopamine shell, and a drug loaded within the system. The polydopamine shell is surface-modified with an FH peptide. The drugs include docetaxel and losartan, with docetaxel loaded on the RGD peptide-modified graphene oxide carrier and losartan loaded in the FH peptide-modified polydopamine shell. This FH peptide-polydopamine-coated graphene oxide drug delivery system, through surface modification of graphene oxide with an RGD peptide and coating with a polydopamine shell, effectively shields against the potential cytotoxicity and blood incompatibility risks of unmodified graphene oxide. Compared to unmodified graphene oxide carriers, this system shows significantly improved compatibility at both normal and tumor cell levels, maintaining extremely high cell viability across a wide concentration range.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and pharmaceutical formulation technology, specifically to the FH peptide-polydopamine-coated graphene oxide drug delivery system and its preparation and application. Background Technology

[0002] The treatment of malignant tumors is one of the major challenges in the current biomedical field, especially in highly fibrotic solid tumors such as pancreatic cancer and breast cancer. The treatment effect has long been limited by the special pathophysiological structure of tumors. These tumors have a dense extracellular matrix barrier inside: cancer-associated fibroblasts (CAFs), as the core matrix component of the tumor microenvironment, secrete a large amount of extracellular matrix components such as collagen and hyaluronic acid. The excessive deposition of these components forms high solid stress, which builds a rigid and densely packed physical barrier around tumor cells. This not only hinders the infiltration of immune cells into the tumor, but also prevents traditional chemotherapy drugs from penetrating deep into the tumor. As a result, most drugs remain at the edge of the tumor matrix and cannot reach the core of the lesion. Ultimately, it is difficult to completely eliminate the tumor tissue, which becomes an important cause of tumor recurrence and drug resistance. Meanwhile, commonly used hydrophobic chemotherapy drugs such as docetaxel also have significant delivery bottlenecks: their water solubility is extremely low, only 0.025 μg / mL, and clinical formulations require solubilizers such as ethanol to assist in dissolution. This not only easily causes adverse reactions such as allergic reactions and fluid retention, but also poses a risk of precipitation after dilution, which greatly limits the dosage and infusion rate. In addition, traditional formulations lack tumor targeting, and the drugs are distributed non-specifically throughout the body after entering the body. While killing tumor cells, they can also cause dose-limiting toxicities such as bone marrow suppression, neurotoxicity, and gastrointestinal reactions, which greatly compresses the treatment window. Furthermore, the free drugs are easily cleared by the body and have a short half-life, making it difficult to maintain an effective therapeutic concentration at the tumor site, which further weakens the anti-tumor effect. Graphene oxide (GO), with its large specific surface area, abundant oxygen-containing functional groups, and excellent photothermal conversion capabilities, has been widely explored for the construction of nanomedicine delivery systems. Existing research has yielded related solutions for GO-based antitumor drug carriers and immune adjuvant / vaccine delivery. However, existing GO-based systems still have many shortcomings: First, insufficiently modified GO poses risks of cytotoxicity and blood compatibility, making it difficult to meet the safety requirements for intravenous administration. Second, drug loading in most systems depends on non-covalent interactions, resulting in poor colloidal and drug loading stability in the complex in vivo fluid environment, which can easily lead to premature drug leakage and uncontrollable in vivo distribution. Third, existing solutions mostly focus on single treatment modalities, achieving only chemotherapy or photothermal therapy, which is difficult to address the multidimensional pathological characteristics of tumors, resulting in limited overall antitumor efficacy. In summary, there is an urgent need to develop a novel nanodelivery system for tumor therapy that can simultaneously achieve stable co-loading of hydrophobic chemotherapeutic drugs and matrix modulators, possess good biocompatibility to adapt to intravenous administration, respond to the tumor microenvironment to achieve precise and controllable drug release, and break through the drug penetration barrier by reshaping the dense tumor matrix, ultimately achieving synergistic effects of chemotherapy, matrix regulation and physical stimulation, and breaking through the bottleneck of existing tumor therapy. Summary of the Invention

[0003] The purpose of this invention is to provide an FH peptide-polydopamine-coated graphene oxide drug delivery system and its preparation and application, in order to solve the problems mentioned in the background art, such as the dense extracellular matrix in highly fibrotic tumors hindering drug penetration, poor stability and large systemic toxic side effects of hydrophobic chemotherapy drugs, insufficient biocompatibility of existing graphene oxide-based carriers, uncontrollable drug release, and the difficulty in achieving synergistic effects with a single treatment modality.

[0004] To achieve the above objectives, the present invention provides the following technical solution: an FH peptide-polydopamine-coated graphene oxide drug delivery system, comprising an RGD peptide-modified graphene oxide carrier, a polydopamine shell, and a drug loaded within the system, wherein the surface of the polydopamine shell is modified with an FH peptide; the drug comprises docetaxel and losartan, wherein docetaxel is loaded on the RGD peptide-modified graphene oxide carrier, and losartan is loaded in the FH peptide-modified polydopamine shell.

[0005] Preferably, the RGD peptide is covalently grafted onto the carboxyl groups on the surface of graphene oxide via an EDC / NHS-mediated amidation reaction. The FH peptide forms covalent bonds with functional groups on the surface of polydopamine via Michael addition / Schiff base reaction, and is non-specifically adsorbed onto the surface of polydopamine via hydrogen bonding and hydrophobic interactions. Docetaxel and losartan are non-covalently loaded into the carrier system through electrostatic interactions, hydrogen bonding, hydrophobic interactions, π-π stacking, and the porous / mesh encapsulation of the polydopamine shell.

[0006] By adopting the above technical solution, the stability of the carrier modification layer is ensured, and the stable co-loading of the two drugs is achieved, avoiding the problems of low drug loading and easy leakage of traditional carriers.

[0007] Preferably, the docetaxel has a drug loading rate of 19.62%; and the losartan has a drug loading rate of 26.43%.

[0008] The above technical solution can provide a sufficient dose basis for both chemotherapy killing and matrix regulation, solving the problems of low delivery efficiency of hydrophobic chemotherapy drugs and difficulty in simultaneous delivery of matrix regulators.

[0009] Preferably, the hydrated particle size of the system is 274.37 nm, the polydispersity index (PDI) is 0.0417, and the surface potential is -8.18 mV.

[0010] By adopting the above technical solution, the narrow particle size distribution and moderate surface potential take into account both colloidal dispersion stability and non-specific adsorption balance, which is suitable for the in vivo circulation requirements of intravenous administration and can avoid rapid clearance or abnormal aggregation of the formulation in vivo.

[0011] Preferably, the system exhibits dual-response release characteristics under both tumor microenvironment acidity and near-infrared light irradiation: after incubation at pH 5.5 for 72 hours, the cumulative release rate of docetaxel is 25.9%, and the cumulative release rate of losartan is 33.2%; under 808nm near-infrared light triggering, after incubation at pH 5.5 for 72 hours, the cumulative release rate of docetaxel is 45.5%, and the cumulative release rate of losartan is 56%, with losartan releasing faster than docetaxel, demonstrating spatiotemporally controllable exposure at the tumor site.

[0012] Using the above technical solution, the drug is firmly locked under normal physiological conditions and released on demand only when triggered by the acidic microenvironment of the tumor or by external light. At the same time, it achieves the synergistic timing of "losartan first releasing the loose matrix and docetaxel later releasing to kill the tumor", which greatly reduces the toxic side effects on normal tissues.

[0013] Preferably, the drug is highly dispersed in the carrier, and XRD detection shows that the crystal diffraction peaks of the free drug are significantly weakened or disappear, indicating that the drug has been successfully loaded into the carrier system.

[0014] By adopting the above technical solution, the problems of free drugs being prone to crystallization and aggregation are avoided, the stability of the formulation storage and in vivo circulation process is improved, and the risk of drug precipitation is reduced.

[0015] Preferably, the system has dual targeting capabilities for both tumor cells and cancer-associated fibroblasts: the RGD peptide can guide the carrier to accumulate in tumor cells, and the FH peptide can specifically recognize the highly expressed TenascinC protein in the tumor microenvironment and deliver it to cancer-associated fibroblasts.

[0016] By adopting the above technical solution, the secretion of matrix can be regulated from the source, breaking through the dense extracellular matrix barrier, and delivering drugs to the deep part of the tumor, thus solving the clinical problem of "difficulty in drug delivery" in solid tumors.

[0017] Preferably, the system has a stable and repeatable photothermal conversion capability, and the heating effect under 808nm near-infrared irradiation is linearly adjustable with laser power density and material concentration, and remains stable after multiple heating / cooling cycles.

[0018] The above-mentioned technical solution can support the combined application of chemotherapy and photothermal therapy, and further improve the tumor ablation effect through the superimposed effect of physical thermotherapy and drug killing.

[0019] Preferably, the system can reduce the mRNA expression levels of α-smooth muscle actin (α-SMA), collagen type I α1 (COL1A1), and collagen type I α2 (COL1A2) in cancer-associated fibroblasts.

[0020] By adopting the above technical solutions, collagen deposition in tumor tissue can be substantially reduced, the dense stroma of highly fibrotic tumors can be loosened, the internal mass transfer environment of tumors can be improved from the root, and physical obstacles can be cleared for subsequent immunotherapy, radiotherapy and other therapies.

[0021] Compared with the prior art, the beneficial effects of the present invention are: the FH peptide-polydopamine coated graphene oxide drug delivery system: 1. Improved biocompatibility and enhanced safety for intravenous administration: Surface modification of graphene oxide with RGD peptides, combined with a polydopamine shell coating, effectively shields against the potential cytotoxicity and blood incompatibility risks associated with raw graphene oxide. Compared to unmodified graphene oxide carriers, this system exhibits significantly improved biocompatibility at both normal and tumor cell levels, maintaining extremely high cell viability across a wide concentration range. This reduces the risks of hemolysis, coagulation, or acute toxicity associated with traditional carrier intravenous administration, significantly increasing the likelihood of clinical translation. 2. Outstanding dual-drug co-loading capacity and superior drug loading stability compared to traditional formulations: This system breaks through the bottleneck of "low drug loading and easy leakage" of hydrophobic chemotherapy drugs and traditional nanocarriers. It can stably load high doses of chemotherapy drugs and matrix modifiers on the same platform at the same time. Compared with the characteristics of free drugs being prone to crystallization and aggregation, the drugs in this system exist in a highly dispersed or amorphous form, which not only significantly improves the drug loading efficiency, but also effectively avoids the risk of drug precipitation during formulation storage and in vivo circulation, providing a solid foundation for stable in vivo delivery. 3. Excellent colloidal stability and more controllable in vivo behavior: The system has ideal uniformity of particle size distribution, which effectively avoids the agglomeration and sedimentation problems common in traditional nano-formulations. Its surface potential design takes into account the balance between dispersion stability and non-specific adsorption, making the formulation less likely to aggregate or be rapidly cleared in complex blood and body fluid environments, and able to maintain a longer blood circulation time, thereby significantly improving the enrichment efficiency of drugs at the tumor site. 4. Intelligent response release for precise drug delivery to tumor sites: Unlike the burst release or uncontrolled release of traditional formulations, this system has dual response characteristics of tumor microenvironment acidity and near-infrared photothermal properties. Under normal physiological conditions, the drug is firmly locked inside the carrier. Once it reaches the acidic tumor microenvironment, it can trigger accelerated drug release. If supplemented with near-infrared light irradiation, it can achieve on-demand release. This "spatiotemporally controllable" release mode not only significantly reduces the toxic side effects of drugs on normal tissues, but also achieves the optimal synergistic logic of "loosening the matrix first and then killing the tumor" through the time difference of matrix modulator release first and chemotherapy drug release later. 5. Dual-targeting mechanism to overcome tumor penetration barrier: The system integrates dual targeting capabilities for tumor cells and cancer-associated fibroblasts. On the one hand, the RGD peptide-guided carrier efficiently accumulates in tumor cells; on the other hand, the FH peptide specifically recognizes and enters fibroblasts in the tumor matrix, regulating matrix secretion from the source. Compared with single-targeting or non-targeting agents, this system can more effectively penetrate the dense extracellular matrix barrier and deliver drugs to the deep tumors that traditional agents cannot reach, solving a common problem in clinical treatment, especially in highly fibrotic solid tumors, where "drugs have difficulty penetrating deep into the tumor." 6. Robust photothermal performance, supporting multimodal synergistic therapy: The system exhibits excellent and stable photothermal conversion capabilities, with the heating effect linearly adjustable according to the intensity of external stimuli, and good reusability stability. This provides a reliable guarantee for the combined application of chemotherapy and photothermal therapy. Through the superimposed effect of physical thermotherapy and drug killing, the tumor ablation effect is further improved. 7. Reshaping the tumor microenvironment to fundamentally reverse drug resistance: Unlike traditional approaches that only focus on directly killing tumor cells, this system substantially reduces collagen deposition in tumor tissue by downregulating the expression of key matrix proteins, significantly loosening the dense stroma of highly fibrotic tumors. This mechanism fundamentally improves the mass transfer environment inside the tumor, not only enhancing the penetration depth of the drugs carried by this system, but also clearing physical barriers for subsequent treatments (such as immunotherapy and radiotherapy), achieving an upgrade from "simple tumor killing" to "remodeling the tumor microenvironment". Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the FH peptide structure of the present invention; Figure 2 The equation for the carboxylation of GO in this invention is as follows; Figure 3 Transmission electron microscopy (TEM) images of graphene oxide, RGD peptide-modified reduced graphene oxide, and polydopamine-encapsulated RGD peptide-modified reduced graphene oxide of the present invention. Figure 4 Scanning electron microscope (SEM) images of graphene oxide-based materials at different modification stages of this invention. Figure 5 This is a schematic diagram illustrating the in vitro drug release behavior of DRGO@FPL under different pH conditions according to the present invention; Figure 6 This is an image showing the staining of live and dead cells in 4T1 cells under different treatments according to the present invention. Figure 7 This is a 3D tumor spheroid permeability observation diagram of the nanocomposite of the present invention. Detailed Implementation

[0023] The technical solutions of 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. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Please see Figures 1-7 The present invention provides a technical solution: FH peptide-polydopamine coated graphene oxide drug delivery system.

[0025] Example 1: Preparation of DRGO@FPL nanocomposite Step 1: Carboxylation of graphene oxide to prepare GO-COOH A certain mass of graphene oxide powder was mixed with deionized water and ultrasonically dispersed for 2 hours to prepare a graphene oxide dispersion with a concentration of 1 mg / mL. Sodium hydroxide was slowly added and stirred under ice-water bath conditions. Chloroacetic acid was then added while stirring, dissolved in water, and added to the graphene oxide dispersion. The mixture was ultrasonically stirred for 8 hours. The mass ratio of chloroacetic acid, sodium hydroxide, and GO was 50:50:1. After the reaction was complete, 1 mol / L dilute hydrochloric acid was added, and the mixture was centrifuged at 14000 r / min for 40 min and washed repeatedly until the pH was neutral, yielding a carboxylated graphene oxide dispersion.

[0026] Step 2: Preparation of RGD peptide-modified graphene oxide (RGO) Carboxylated graphene oxide dispersion was added to EDC and NHS as coupling agents to form amide bonds between the amino groups of RGD peptide and the carboxyl groups on the GO surface. The molar ratio of EDC to NHS was 1.2:1. The pH was adjusted to 6, and the mixture was stirred at 250 rpm for 30 minutes to activate the carboxyl groups. The pH was then adjusted to 8, and an aqueous solution of RGD peptide was added to the activated graphene oxide dispersion. The reaction was carried out overnight at room temperature with stirring. After the reaction, the system was washed, dialyzed, and lyophilized to obtain RGO powder. The reaction conditions were: a GO to RGD peptide mass ratio of 10:1. The reaction was carried out at room temperature with stirring for approximately 24 hours, followed by repeated washing. The mixture was dialyzed in deionized water using a 3.5 kDa molecular weight cutoff dialysis bag for approximately 72 hours, and then lyophilized to obtain RGD peptide-modified graphene oxide carrier RGO.

[0027] Step 3: Preparation of docetaxel-loaded DRGO Docetaxel was dissolved in dimethyl sulfoxide to obtain a drug stock solution. This stock solution was added to an RGO aqueous solution at a predetermined mass ratio. The reaction was carried out under light-protected and stirred conditions, allowing the drug to be adsorbed / bound to the RGO surface or between its layers via non-covalent interactions. After the reaction, the system was dialyzed and lyophilized to obtain the RGD peptide-modified graphene oxide-based nanoparticle drug delivery system DRGO. The reaction conditions were: RGO to DTX mass ratio 1:1, stirring at room temperature in the dark for approximately 72 hours, followed by dialyzing in deionized water using an 8-14 kDa dialysis bag for approximately 72 hours to remove free drug.

[0028] Step 4: Preparation of polydopamine-coated DRGO@P 10 mg DRGO was dispersed in 10 mL of pH 8.5, 10 mM Tris-HCl buffer and sonicated for 5 min to obtain a uniform dispersion. Then, 10 mg of dopamine hydrochloride was added, and the black solution was stirred at 600 rpm for 12 hours at room temperature in the dark. As the reaction between PDA and GO proceeded, the color of the mixture gradually changed from the initial brown of GO to black. The concentrations of PDA and GO were both 1.0 mg / mL. The mixture was centrifuged in deionized water and ethanol at 8000 rpm for 20 minutes and washed repeatedly until the supernatant became colorless. The mixture was then lyophilized to obtain the polydopamine-coated RGD peptide-modified graphene oxide-based nanocarrier system DRGO@P.

[0029] Step 5: Preparation of FH peptide-modified and losartan-loaded DRGO@FPL DRGO@P was resuspended in an aqueous solution containing FH peptide. After stirring at 600 rpm for 12 hours at room temperature, the mixture was centrifuged at 8000 rpm for 20 minutes and washed three times with deionized water to obtain the FH peptide-modified polydopamine-coated RGD peptide-modified graphene oxide-based nanopeptide drug delivery system DRGO@FP. The mass ratio of DRGO@P to FH peptide was 2.5:1.

[0030] LOS and DRGO@FP were then mixed in an aqueous solution and stirred for 24 hours. The mixture was then washed three times by centrifugation at 8000 rpm for 20 minutes with deionized water. This yielded DRGO@FPL, a polydopamine-coated RGD peptide-modified graphene oxide-based nanoparticle dual-drug delivery system modified with FH peptide. The mass ratio of DRGO@FP to LOS was 1:1.

[0031] Example 2: In vitro cytotoxicity evaluation Logarithmic growth phase 4T1 cells were seeded at an appropriate density in 96-well culture plates and cultured in an incubator until the cells adhered. Different concentrations of the test sample were added, and after further culture for the corresponding time, CCK-8 reagent was added to each well. The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader, and the cell viability of each group was calculated.

[0032] The results showed that at a lower concentration of 10 ng / mL, the cell viability of the DRGO@FPL+NIR group decreased to 52.2±2.55%, significantly lower than that of the DTX group (94.5±1.48%) and the DRGO@FPL group (78.1±3.47%). This indicates that after modification and combined with photothermal therapy, DRGO@FPL can achieve superior tumor cell killing efficiency at a lower dose. Under conditions of 0-100 μg / mL and 24 h, the overall cell viability of both blank vectors RGO and FPL remained at ≥85%, indicating that the blank vector showed high cell compatibility.

[0033] Example 3: Verification of killing effect by cell live / dead staining 4T1 cells in good growth condition were used at a rate of 4 × 10⁻⁶ 4 MDA-MB-231 cells were seeded at a density of 5 × 10⁶ wells. 4 Cells were seeded at the specified density into 24-well plates and cultured overnight. The old culture medium was discarded, and cells were treated with complete culture medium containing different concentrations of DTX, DGO, DRGO, DRGO@FPL, and DRGO@FPL+NIR. The photothermal therapy group was irradiated with an 808 nm laser (1.0 W / cm², 10 min), while the control group received no treatment. Cells were incubated at 37°C for 24 h. Calcein AM / PI working solution was prepared at a ratio of 1000:1:1 (diluent, Calcein AM, and PI), and protected from light. The old culture medium was discarded, and cells were washed twice with pre-cooled PBS. 100 μL of Calcein AM / PI working solution was added to each well, and staining was performed for 20 min, all in the dark. Imaging was performed under a confocal microscope.

[0034] The results are as follows Figure 5As observed, the GO group showed almost entirely green fluorescence with only a very small amount of red fluorescence, indicating that the cell survival rate was high and there was almost no cell death. In contrast, the DRGO@FPL combined with photothermal therapy group showed predominantly red fluorescence with almost no green fluorescence, indicating that a large number of cells died in this group, with a significantly higher number of deaths compared to the DTX group.

[0035] Example 4: Evaluation of 3D Tumor Spheroid Permeability A 1:1 mixture of healthy 4T1 cells and NIH3T3 cells transformed into CAFs by TGF-β stimulation was seeded at a density of 5000 cells / well in low-adsorption 96-well plates and cultured for 5 days to form uniform 3D tumor multicellular spheroids of consistent size. The tumor spheroids were then transferred to different groups of culture media labeled with coumarin 6 (C6) and incubated for the same amount of time. Fluorescence signal distribution images were acquired by scanning layer by layer along the diameter of the spheroids using a laser confocal microscope (CLSM) or a high-content imaging system.

[0036] The results are as follows Figure 7 As shown, the fluorescence signal of the coumarin 6 (C6) group was limited to the edge region of the tumor spheroid, with no obvious fluorescence in the core region, indicating that the formulation could only adhere to the surface of the spheroid and could not effectively penetrate into the interior. The fluorescence signal of the C6+LOS group extended from the edge to the core region, with scattered fluorescence distribution inside the spheroid, but the fluorescence intensity in the core region was still significantly weaker than that at the edge, indicating that the formulation had a certain penetration ability, but the depth and uniformity were insufficient. The light signal of the C6-labeled nano-drug delivery system C6-RGO@FPL group was uniformly distributed throughout the entire tumor spheroid, with no significant difference in fluorescence intensity between the edge and core regions, indicating that the formulation could efficiently penetrate the dense matrix of the tumor spheroid and achieve uniform penetration throughout the entire layer.

[0037] Example 5: Validation of Tumor Stromal Remodeling Effect Further rt-qPCR experiments were used to detect the expression of matrix-related proteins. NIH3T3 cells in good growth condition were subjected to 4×10⁻⁶... 4 Cells were seeded at a density of / wells and stimulated with TGF-β for 24 h to induce cancer-associated fibroblasts (CAFs). Following this, LOS and FPL were added according to groupings and treated for 48 h. After treatment, the culture medium was discarded, and amplification and detection were performed using real-time quantitative PCR. GAPDH internal reference genes were used, and the detected genes included α-smooth muscle actin (α-SMA), collagen type I α1 chain (COL1A1), and collagen type I α2 chain (COL1A2).

[0038] The results showed that the nanocomposite could reduce the mRNA levels of α-smooth muscle actin (α-SMA), collagen type I α1 (COL1A1), and collagen type I α2 (COL1A2), indicating that it could effectively reduce the collagen content in the extracellular matrix of tumor cells, thereby loosening the matrix.

[0039] Example 6: Validation of Dual-Response Drug Release Characteristics DRGO@FPL was placed in release media with different pH conditions, and its release behavior was monitored under a constant temperature of 37°C. At the same time, the light group was triggered by 808nm near-infrared light, and the cumulative drug release under different conditions was compared.

[0040] The results showed that under pH 5.5 conditions, the cumulative release of DTX and LOS reached approximately 25.9% and 33.2% after 72 hours, respectively. Under near-infrared light triggering (808nm) conditions, the cumulative release under pH 5.5 conditions and 72 hours could be further increased to approximately 45.5% for DTX and approximately 56% for LOS. Moreover, losartan was released faster than docetaxel, exhibiting the characteristics of spatiotemporally controllable exposure at the tumor site.

[0041] Figure 1 The image shows the structure of FH peptide. FH peptide is a synthetically produced functional small molecule polypeptide that can specifically bind to target molecules highly expressed in the tumor microenvironment (such as Tenascin C protein, which is mainly secreted by tumor-associated fibroblasts and is abnormally highly expressed in various tumor tissues such as breast cancer, while being lowly expressed or not expressed in normal tissues). It can perform targeted recognition and targeted delivery functions and is an important functional modification fragment in the construction of tumor-targeted nanomedicine carriers. In tumor-targeted therapy, FH peptide can endow nanocarriers with targeting properties to tumor tissues and tumor-associated fibroblasts, help the carrier penetrate the dense matrix barrier of the tumor, reduce non-specific adsorption to normal tissues, improve the enrichment efficiency of nanomedicines at the tumor site, and thus enhance the anti-tumor efficacy of nanomedicines.

[0042] Figure 2 The equation for the carboxylation of GO is shown. By converting the OH groups on the GO sheet into COOH groups, more carboxylic acid functional groups that can be used for subsequent coupling are introduced into the GO, thus providing more sites for subsequent covalent linkage with RGD peptide. The next step is the reaction equation for the amidation coupling of RGD peptide and graphene oxide in an EDC / NHS activation system. Specifically, EDC / NHS first activates the carboxyl groups on the GO surface to form a reactive intermediate, which then undergoes nucleophilic substitution with the primary amine on the RGD peptide molecule to form an amide bond, thereby achieving covalent grafting and obtaining RGD peptide-functionalized graphene oxide support (RGO). This covalent linkage method is beneficial to improving the stability of the modified layer and ensuring the structural integrity of the material under physiological conditions.

[0043] Figure 3 To characterize graphene oxide (GO), drug-loaded graphene oxide modified with RGD peptide (DRGO), and drug-loaded graphene oxide modified with RGD peptide encapsulated with FH peptide-modified polydopamine (PDA) (DRGO@FPL) by transmission electron microscopy (TEM). GO: It exhibits a typical sheet-like structure with a smooth surface, clear edges, and wrinkled morphology characteristic of graphene oxide, without any other modifiers attached; DRGO: After modification with RGD peptide and drug loading, the sheets showed obvious stacking and aggregation, with increased surface wrinkles and rough texture, indicating that the peptide grafting affected the spatial structure of the GO sheets. DRGO@FPL: After being coated with polydopamine, the edges of the sheets show a continuous and uniform dark coating layer. The overall structure is dense and the boundaries are clear, confirming that the polydopamine shell has been successfully coated on the surface of DRGO, forming a core-shell structure.

[0044] Figure 4 Scanning electron microscopy (SEM) images were used to characterize graphene oxide-based materials at different modification stages. The results showed that the surface morphology of the materials evolved regularly with the increase of modification degree. GO: exhibited a typical smooth sheet-like structure with a flat surface, few wrinkles, and relatively loose arrangement between sheets, which is the characteristic morphology of unmodified graphene oxide. With the increase of modification, the degree of sheet stacking was further increased, and the surface texture became rough and uneven. Finally, in the DRGO@FPL group, the sheet structure was completely covered, exhibiting a typical core-shell composite morphology.

[0045] Figure 5 The in vitro drug release behavior of DRGO@FPL under different pH conditions is shown; The left figure shows the DTX release curve, and the right figure shows the LOS release curve. These release curves are used to compare the release differences of the nanocomposite in neutral and acidic environments, thereby simulating the drug release characteristics under acidic compartment conditions such as blood / normal tissue and tumor microenvironment or intracellular endosomes / lysosomes. The results show that the nanocomposite of the present invention exhibits distinguishable release kinetics under different pH conditions and near-infrared irradiation, reflecting its environmentally responsive release trend. This is beneficial to improving the availability of drugs in tumor-associated acidic microenvironments and reducing exposure to non-target sites. Moreover, DTX is released more slowly than LOS, effectively prolonging the drug action time.

[0046] Figure 6 Observation of live and dead cell staining of 4T1 cells under different treatments The control group showed almost no dead cells. Compared with the control group, all drug-treated groups showed varying degrees of increased cell death and decreased proportion of live cells. Among them, the DTX group showed a certain killing effect and a decrease in the proportion of live cells; while the DRGO@FPL+NIR group showed a significant cell-killing effect, which was significantly better than the other groups.

[0047] Figure 7 Observation of the permeability of 3D tumor spheres of nanocomposite The fluorescence signal of the free C6 group was mainly distributed in the peripheral region of the tumor spheroid. As the scanning depth increased from 0 μm to 100 μm, the fluorescence signal inside the spheroid significantly weakened, suggesting that the free fluorescence could not effectively penetrate the dense three-dimensional tumor spheroid structure. The C6+LOS group showed a certain degree of enhanced internal fluorescence compared to the free C6 group, indicating that losartan can improve the internal permeability environment of the tumor spheroid to some extent, but its fluorescence distribution still showed a phenomenon of strong fluorescence at the periphery and weak fluorescence at the center. In contrast, the C6-labeled nanocomposite group showed strong and relatively uniform green fluorescence signals at different scanning depths, and the central region of the spheroid still maintained obvious fluorescence distribution; the three-dimensional reconstruction image on the right further shows that the fluorescence signal of this group was distributed in a more continuous, three-dimensional and uniform manner inside the tumor spheroid.

[0048] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An FH peptide-polydopamine-coated graphene oxide drug delivery system, characterized in that: include: The system comprises an RGD peptide-modified graphene oxide carrier, a polydopamine shell, and a drug loaded within the system, wherein the surface of the polydopamine shell is modified with an FH peptide; the drug comprises docetaxel and losartan, wherein docetaxel is loaded on the RGD peptide-modified graphene oxide carrier, and losartan is loaded in the FH peptide-modified polydopamine shell.

2. The FH peptide-polydopamine-coated graphene oxide drug delivery system according to claim 1, characterized in that: The RGD peptide is covalently grafted onto the carboxyl groups on the surface of graphene oxide via an EDC / NHS-mediated amidation reaction. The FH peptide forms covalent bonds with functional groups on the surface of polydopamine via Michael addition / Schiff base reaction, and is non-specifically adsorbed onto the surface of polydopamine via hydrogen bonding and hydrophobic interactions. Docetaxel and losartan are non-covalently loaded into the carrier system through electrostatic interactions, hydrogen bonding, hydrophobic interactions, π-π stacking, and the porous / mesh encapsulation of the polydopamine shell.

3. The FH peptide-polydopamine-coated graphene oxide drug delivery system according to claim 1, characterized in that: The loading rate of docetaxel was 19.62%; the loading rate of losartan was 26.43%.

4. The FH peptide-polydopamine-coated graphene oxide drug delivery system according to claim 1, characterized in that: The system has a hydrated particle size of 274.37 nm, a polydispersity index (PDI) of 0.0417, and a surface potential of -8.18 mV.

5. The FH peptide-polydopamine-coated graphene oxide drug delivery system according to claim 1, characterized in that: The system exhibits dual-response release characteristics under both tumor microenvironment acidity and near-infrared light irradiation: after incubation at pH 5.5 for 72 hours, the cumulative release rate of docetaxel is 25.9% and the cumulative release rate of losartan is 33.2%; under 808nm near-infrared light triggering, after incubation at pH 5.5 for 72 hours, the cumulative release rate of docetaxel is 45.5% and the cumulative release rate of losartan is 56%.

6. The method for preparing the FH peptide-polydopamine-coated graphene oxide drug delivery system according to any one of claims 1-5, characterized in that: Includes the following steps: Step 1: Carboxylation of graphene oxide: Graphene oxide is reacted with sodium hydroxide and chloroacetic acid, neutralized with hydrochloric acid, and then centrifuged and washed to obtain carboxylated graphene oxide GO-COOH; Step 2: Preparation of RGD peptide-modified graphene oxide (RGO): GO-COOH and RGD peptide were reacted at room temperature in the presence of EDC / NHS with stirring. After dialyzing and lyophilization, RGO powder was obtained. Step 3: Preparation of RGO loaded with docetaxel to obtain DRGO: RGO and docetaxel were stirred in the dark, dialyzed to remove free drug, and then freeze-dried to obtain DRGO; Step 4: Preparation of polydopamine-coated DRGO to obtain DRGO@P: Disperse DRGO in Tris buffer, add dopamine hydrochloride, stir at room temperature in the dark, centrifuge, wash, and freeze dry to obtain DRGO@P; Step 5: Preparation of FH peptide-modified DRGO@FP and the final system DRGO@FPL loaded with losartan: Mix DRGO@P with FH peptide aqueous solution, stir, centrifuge and wash to obtain DRGO@FP; then mix DRGO@FP with losartan aqueous solution, stir to remove free drug and freeze dry to obtain DRGO@FPL.

7. The preparation method of the FH peptide-polydopamine-coated graphene oxide drug delivery system according to claim 5, characterized in that: The carboxylation reaction conditions for graphene oxide in step 1 are as follows: after ultrasonic dispersion of graphene oxide, it is mixed with sodium hydroxide and chloroacetic acid aqueous solution and ultrasonicated for 2 hours, neutralized with 1 mol / L hydrochloric acid, centrifuged at 14000 rpm for 40 minutes, and the precipitate is washed until neutral and then resuspended in deionized water. In step 2, the concentration of GO-COOH was 2 mg / mL, the concentration of RGD peptide was 0.2 mg / mL, the reaction was stirred at room temperature for 24 h, and dialyzed for 72 h using a 3.5 kDa molecular weight cutoff dialysis bag. In step 3, the concentration of RGO is 1 mg / mL, the concentration of docetaxel is 1 mg / mL, the mixture is stirred in the dark for 72 h, and dialysis is performed using an 8-14 kDa dialysis bag for 72 h. In step 4, the Tris buffer was pH 8.5, 10 mM Tris-HCl buffer, the DRGO concentration was 1.0 mg / mL, the dopamine hydrochloride concentration was 1.0 mg / mL, and the mixture was stirred at 600 rpm for 24 h at room temperature in the dark, followed by centrifugation at 8000 rpm for 20 min. In step 5, the concentration of DRGO@PDA was 2.5 mg / mL and the concentration of FH peptide was 1.0 mg / mL. The mixture was stirred for 24 h, centrifuged and washed, and then DRGO@FP was mixed with 1 mg / mL losartan aqueous solution at a volume ratio of 1:1 and stirred for 24 h.

8. The application of the FH peptide-polydopamine-coated graphene oxide drug delivery system according to any one of claims 1-5, characterized in that: The system is used for tumor treatment, and is particularly suitable for scenarios where chemotherapy is required to kill tumor cells and loosen the extracellular matrix of tumor cells simultaneously. It can be administered via intratumoral injection or intravenous injection.

Images