CN114404610A - Hyaluronic acid-geraniol polymer prodrug multi-biological response drug delivery system HSSG NPs and preparation method and application thereof - Google Patents

Hyaluronic acid-geraniol polymer prodrug multi-biological response drug delivery system HSSG NPs and preparation method and application thereof Download PDF

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CN114404610A
CN114404610A CN202210100124.1A CN202210100124A CN114404610A CN 114404610 A CN114404610 A CN 114404610A CN 202210100124 A CN202210100124 A CN 202210100124A CN 114404610 A CN114404610 A CN 114404610A
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段少峰
岑娟
夏一帆
白银都
崔杰
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Abstract

The invention belongs to the field of biological pharmacy, relates to construction of a polymer prodrug multi-biological response drug delivery system, and particularly relates to hyaluronic acid-geraniol polymer prodrug multi-biological response drug delivery system HSSG NPs, and a preparation method and application thereof. By binding GER to HA using disulfide bonds, a simple redox sensitive GER hyaluronic acid polymer prodrug is synthesized. The synthesized prodrug HSSG as a molecular structural motif can self-assemble into simple and multifunctional nanoparticle HSSG NPs in aqueous solution. The NPs exhibit accelerated drug release rates when exposed to buffers that mimic the multiple features in the tumor microenvironment. HSSG NPs remain stable in different physiological media. Compared with GER and HCCG NPs, HSSG NPs significantly promote cancer cell death and enhance inhibition of tumor growth with reduced toxicity.

Description

Hyaluronic acid-geraniol polymer prodrug multi-biological response drug delivery system HSSG NPs and preparation method and application thereof
Technical Field
The invention belongs to the field of biological pharmacy, relates to construction of a polymer prodrug multi-biological response drug delivery system, and particularly relates to hyaluronic acid-geraniol polymer prodrug multi-biological response drug delivery system HSSG NPs, and a preparation method and application thereof.
Background
Liver cancer is the third leading cause of cancer-related death worldwide. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for 75-85% of all cases. The 5-year survival rate of HCC patients is only 18%, making them the second-most life malignancy next to pancreatic cancer. Chemotherapy, in addition to surgery and radiation therapy, remains the primary treatment for neoplastic disease. Although various chemotherapeutic drugs including sorafenib, lovastatin, doxorubicin, and mitoxantrone are used in clinical treatment of liver cancer as single drugs or combined drugs, they have poor tissue selectivity, cannot effectively target tumor sites, limit therapeutic effects, and cause serious side effects. There is therefore a need for effective drugs with high efficacy and low toxicity for the treatment of HCC. Natural compounds can be used to create new therapies.
Geraniol is a naturally occurring acyclic monoterpene found in geranium, citronella, litsea cubeba of the family lauraceae, and rosaceous. It has a wide range of pharmacological properties including antioxidant, antimicrobial, anti-inflammatory, neuroprotective and antiulcer properties, as well as insect repellent and insecticide properties. Meanwhile, it has been demonstrated to have antitumor effects on liver cancer, melanoma, endometrial cancer, colon cancer cells, prostate cancer and tongue cancer in vitro and in vivo, as well as to play a role in preneoplastic lesions. However, it is difficult to exert a beneficial effect in clinical treatment due to its poor solubility, volatility and bioavailability. Self-assembling nano-polymers have attracted a great deal of attention in the field of cancer treatment in the past decade because of their ability to significantly increase the solubility of hydrophobic drugs, prolong blood circulation, and improve anticancer efficacy. Recent studies have shown that when specific factors in the tumor microenvironment are activated, bioresponsive nanoparticles may change their physicochemical properties, leading to spatial release of the drug in the tumor cells. Since many biological factors are changed at the tumor site at the same time, multi-bioresponse nanoparticles have been demonstrated to exhibit better tumor-specific drug release capacity than a single stimulus response. It is well known that Hyaluronic Acid (HA) is widely used in the biomedical field due to its high biocompatibility, immunogenicity and hydrophilicity. In addition, HA can specifically bind to CD44 receptor overexpressed by a variety of cancer cells, and thus can be used as a drug targeting vector for drug delivery and cancer treatment. In addition, HA, as a natural ligand for CD44, can also be specifically degraded by hyaluronidase (HAase) at the tumor site.
Intracellular concentrations of the redox agent Glutathione (GSH) are reported to be as high as 10mM, while their concentration in extracellular fluid is only 2-20. mu.M. In addition, the content of GSH in cancer cells is four times that of normal cells. Therefore, the drug conjugate or nanoparticle containing a disulfide bond can be decomposed by adding glutathione, thereby achieving a redox-reactive drug release effect.
Disclosure of Invention
The invention provides a hyaluronic acid-geraniol polymer prodrug multi-biological response drug delivery system HSSG NPs and a preparation method and application thereof, wherein the drug delivery system can respond to pH, GSH or HAase, and solves the technical problems that the existing Geraniol (GER) is low in water solubility and poor in bioavailability, and the clinical conversion of the geraniol is greatly hindered.
The technical scheme of the invention is realized as follows:
a hyaluronic acid-geraniol polymer prodrug multi-bioresponse drug delivery system HSSG NPs having the structure:
Figure 100002_DEST_PATH_IMAGE001
。
further, the delivery system HSSG NPs are synthesized by disulfide bonding and self-assembly of hydrophilic group HA and hydrophobic group GER on HSSG NPs.
Further, the delivery system HSSG NPs have pH/glutathione/hyaluronidase responsive drug release capacity.
The synthetic route of the above drug delivery system HSSG NPs is as follows:
Figure DEST_PATH_IMAGE002
the preparation method comprises the following steps:
(1) HA (300 mg, 0.792 mmol), EDC & HCl (0.5-2 mmol) and NHS (109.3 mg, 0.5-2 mmol) were dissolved in 10 mL Deionized (DI) water with stirring at 25 ℃ to activate the carboxyl groups of HA. After stirring for 30 min, CYS (535.1 mg, 1-5 mmol) was added to the above activated NHS ester of HA and the reaction mixture was stirred for about 24 h. Thereafter, the reaction mixture was poured into a dialysis tube (MWCO 3.5kDa) and dialyzed for 72 hours. Finally, the product HA-CYS was collected by lyophilization.
(2) GER (1.54 g, 10 mmol), SA (5-15 mmol) and DMAP (10 mg, catalytic amount) were weighed accurately into a 200ml dry round bottom and 50ml dry dichloromethane were added. Adding 0.001-0.02 mmol of pyridine under the protection of argon, and stirring at room temperature for 24 hours. After the reaction is finished, 2N hydrochloric acid and H are sequentially used2O wash and organic phase dried over magnesium sulfate. After removal of the solvent, the product GER-SA is obtained as a yellow oil.
(3) HA-CYS (476mg) was dissolved in 3 mL formamide under mild heating and cooled to 25 ℃. Then, the solution was diluted with 2mL of DMSO. GER-SA (40-80 mg), EDC (40-80 mg) and NHS (20-50 mg) were dissolved in 5mL DMF at 25 ℃. After stirring for 30 minutes, the HA-CYS solution was introduced into the activated GER solution and then stirred at 25 ℃ for 24 hours. The final mixture was dialyzed for 72 hours (MWCO 3.5 kDa). HA-SS-GER (HSSG) was collected by lyophilization.
(4) HSSG was sonicated at 0 ℃ and 150W with a probe sonicator and then dispersed in 5mL deionized water, and the sonicated dispersion was filtered through a 0.45 μm membrane filter to obtain HCCG NPs.
The application of the HSSG NPs of the drug delivery system in preparing a drug targeting CD44 receptor.
The application of the drug delivery system HSSG NPs in preparing antitumor drugs is that the usage size of the drug delivery system HSSG NPs is 101.7nm, and the drug loading efficiency is 18.5%.
The invention has the following beneficial effects:
1. the targeted and multiple bioresponsive HSSG NPs of the invention are useful for active CD44 receptor mediated selective GER delivery, multiple bioresponsive rapid GER release, which results in enhanced intracellular accumulation of GERs to induce cancer cell apoptosis and enhance anticancer activity. In addition, HCCG prodrug conjugates link HA and GER through adipic Acid Dihydrazide (ADH) linker as an unreactive control with amide bond formation.
2. The multiple bioresponse drug delivery system HSSG NPs of the invention have the required particle size of only 107.1 nm, have an average diameter of about 110 nm, are uniformly spherical and maintain stability in different physiological media. Furthermore, when these NPs were exposed to a buffer mimicking the multiple features (pH/glutathione/hyaluronidase) in the tumor microenvironment, they showed accelerated drug release rates. The results of fluorescence microscopy and flow cytometry confirm that the nanosystem is selectively taken up by the human hepatoma cell lines HepG2 and Huh7 by targeting the internalization mediated by the CD44 receptor. And drug release capacity in response to pH/glutathione/hyaluronidase.
3. The multi-biological response drug delivery system HSSG NPs have high stability, and the internalization efficiency of the HA-containing NPs in hepatoma cell HepG2, Huh7 and H22 tumor-bearing mice is improved through the functionalization of the HA-containing NPs. Compared with GER and HCCG NPs, the cancer cell death is promoted remarkably and the inhibition of tumor growth is enhanced, and the toxicity is reduced. Therefore, HSSG NPs have great potential as a simple and efficient platform for tumor-targeted drug delivery and therapy.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Figure 1 is a synthesis and characterization of HSSG prodrug conjugates. Wherein (A) the synthetic route of the HSSG conjugate. (B) D2HA and CDCl in O3Middle GER, D2HCCG and D in O2Of HSSG in O1H NMR spectrum. (C) FT-IR spectra of HA, GER, HCCG and HSSG.
Fig. 2 is a preparation and characterization of HSSG and HSSG NPs, where (a) TEM images of HCCG (left) and HSSG (right) [ scale bar: 500 nm ]. (B) Particle size distribution of HCCG and HSSG. (C) Time-dependent particle size of HCCG and HSSG in 0.1M PBS (pH 7.4) and 0.1M PBS (containing 10% FBS). (D) With different pH values (pH = 5.5, 6.8, 7.4). (E) In vitro cumulative release profiles of drug from HA functionalized NPs at different GSH concentrations (0, 10 μ M and 10 mM) in buffer pH = 6.8 and (F) various HAase concentrations at pH = 6.8 (0, 5 and 50 IUmL-1). Data are expressed as mean ± s.e.m. (n = 3).
FIG. 3 shows cellular uptake of IR 780-HSSG. Fluorescence images of (a) HepG2 and (B) Huh-7 cells interacting with IR780-HSSG at different times (0, 1,3, 6, 12 and 24h), red fluorescence indicating the presence of IR780-HSSG and blue fluorescence indicating DAPI positive staining, [ scale bar = 100 μm ]. (C) Flow cytometric analysis of the interaction of HepG2 and (D) Huh-7 cells with IR780-HSSG at different times (0, 1,3, 6, 12 and 24 hours) and the mean fluorescence intensity of IR780-HSSG was calculated. (E) Flow cytometry analysis of HepG2 and (F) Huh-7 cells interacted with IR780-HSSG and various uptake inhibitors. The mean fluorescence intensity of IR780-HSSG was calculated. Data are expressed as mean ± s.e.m. (n = 3, # P < 0.05, # P < 0.001).
Figure 4 is an in vitro cellular uptake study. Wherein (A) HA, GER, HCCG and HSSG cytotoxicity against HepG2 and Huh7 cells at 24, 48 and 72 h. (B) Induced HepG2 and Huh7 cell viability was treated with different preparations of Calcein-AM/PI staining. [ scale bar = 200 μm ].
FIG. 5 shows the effect of inhibiting proliferation and causing cell cycle arrest and apoptosis in vitro. (A) The EdU method examined the DNA replication activity of each group of HepG2 and Huh-7 cells, [ scale bar = 100 μm ]. (B) determination of apoptosis levels by TUNEL method, [ scale bar = 100 μm ]. (C) Proliferation rates were analyzed for each group. (D) The apoptosis index is calculated by the formula: apoptosis index = (positively stained apoptotic cells)/(total number of cells) × 100%. (E) the rate of apoptosis was measured using flow cytometry on HepG2 and Huh-7 cells treated with PBS, HA, GER, HCCG and HSSG. (F) Flow cytometry assays were used to detect cell cycle distribution in HepG2 and Huh7 cells. (G) The percentage (%) of apoptotic cells was calculated. (H) Statistical analysis of cell cycle results. (I) Mitochondrial membrane potential fluorescence image detected by JC-1 method. [ scale bar = 50 μm ]. (J) Western blot analysis of expression levels of apoptotic proteins. (K, L) densitometric analysis of each factor in each group was normalized to the corresponding GAPDH level. Data are expressed as mean ± s.e.m. (n = 3, # P < 0.05, # P < 0.01, # P < 0.001 as compared to the control group, # P < 0.05, # P < 0.01, # P < 0.001 as compared to the HSSG group).
FIG. 6 is an in vivo biodistribution map. (A) Optical photographs of tumor tissues excised from the host two weeks after treatment of H22 tumor-bearing mice with saline, HA, GER, HCCH and HSSG. (B) changes in tumor volume during treatment. (C) weight of tumor tissue at day 15. (D) H & E staining, Ki-67 staining and TUNEL staining of tumor sections from different treatment groups. [ scale bar = 50 μm ] (E) in vivo fluorescence images of H22 tumor-bearing mice at 1,3, 6, 12 and 24 hours after injection of IR 780-HSSG. (F) fluorescence images of five major organs and tumors 24h after intravenous injection of IR-780-HSSG. Data are expressed as mean ± s.e.m. (n = 6). P < 0.05, P < 0.005; in comparison to the HSSG group, # # P < 0.01, # # P < 0.001.
FIG. 7 is a graph of in vivo anti-tumor efficacy. (A) H22 tumor-bearing mice changed body weight after two weeks of intravenous injection of saline, HA, GER, HCCG and HSSG. (B) representative images of tumors and tumor weights harvested on day 15 for each treatment group. (C) Histogram of liver and kidney function indices: ALT, AST, UREA and CREA. (D) typical HE stained histological images obtained from liver, spleen, lung, kidney and heart. [ scale bar = 50 μm ].
Figure 8 is a synthetic route to HCCG prodrug conjugates.
FIG. 9 is a graph showing the results of a hemolysis experiment.
FIG. 10 is a graph showing zeta potential results of HCCG NPs and HSSG NPs.
Detailed Description
The main reagents, drugs and samples used in this application:
HA (Mw = 8 kDa) was purchased from Bloomage Freda Biopharm co., Ltd. (shandong, china). 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl), 1-hydroxypyrrolidine-2, 5-dione (NHS), adipic Acid Dihydrazide (ADH), cystamine dihydrochloride (CYS), Geraniol (GER), Succinic Anhydride (SA), 4-Dimethylaminopyridine (DMAP), 2- [2- [2-chloro-3- [ (1,3-dihydro-3, 3-dimethyl-1-propyl-2H-indol-2-ylidine) ethylidene ] -1-cyclohexen-1-yl ] vinyl ] -3, 3-dimethyl-1-propyliodoindole (IR780) and 3- (4, 5-dimethylthiazol-2-yl) -2, 5-Diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, Mo., USA). Other organic solvents and reagents were purchased from the national pharmaceutical group chemical reagents, ltd. Fetal Bovine Serum (FBS) was purchased from GIBCO. Trypsin and high-sugar Dulbecco's Modified Eagle Medium (DMEM) were purchased from Beijing sunlight Biotechnology Ltd (Beijing, China). All reagents and solvents were used directly.
Experimental Swiss mice (26. + -.2 g) at 6 weeks of age were purchased from the Experimental animals center in Henan (Zheng, China).
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
The preparation method of HSSG of the present embodiment is shown in fig. 1, and comprises the following specific steps:
(1) HA (300 mg, 0.792 mmol), EDC & HCl (182 mg, 0.950 mmol) and NHS (109.3 mg, 0.950 mmol) were dissolved in 10 mL Deionized (DI) water with stirring at 25 ℃ to activate the carboxyl groups of HA. After stirring for 30 min, CYS (535.1 mg, 2.376 mmol) was added to the above activated NHS ester of HA and the reaction mixture was stirred for about 24 h. Thereafter, the reaction mixture was poured into a dialysis tube (MWCO 3.5kDa) and dialyzed for 72 hours. Finally, the product HA-CYS was collected by lyophilization.
(2) GER (1.54 g, 10 mmol), SA (1.20 g, 12 mmol) and DMAP (10 mg, catalytic amount) were weighed out accurately in a 200ml dry round bottom and 50ml dry dichloromethane were added. 0.791g of pyridine was added under argon and stirred at room temperature for 24 hours. After the reaction is finished, 2N hydrochloric acid and H are sequentially used2O wash and organic phase dried over magnesium sulfate. After removal of the solvent, the product GER-SA is obtained as a yellow oil.
(3) HA-CYS (476mg) was dissolved in 3 mL formamide under mild heating and cooled to 25 ℃. Then, the solution was diluted with 2mL of DMSO. GER-SA (60 mg, 0.132 mmol), EDC (60.8 mg, 0.396 mmol) and NHS (36.5 mg, 0.396 mmol) were dissolved in 5mL DMF at 25 ℃. After stirring for 30 minutes, the HA-CYS solution was introduced into the activated GER solution and then stirred at 25 ℃ for 24 hours. The final mixture was dialyzed for 72 hours (MWCO 3.5 kDa). HA-SS-GER (HSSG) was collected by lyophilization.
The comparative HA-ADH-GER (HCCG) conjugate was prepared using the same procedure as that used in example 1, except that CYS was replaced with ADH.
(4) Preparation of HCCG NPs and HSSG NPs: and (3) placing the HSSG prodrug/HCSG prodrug at 0 ℃ and 150W, performing ultrasonic treatment by a probe ultrasonic instrument, dispersing in 5mL of deionized water, and filtering the obtained ultrasonic dispersion liquid by a 0.45-micrometer membrane filter to obtain HCCG NPs and HSSG NPs.
And structural characterization of HCCG conjugateRecording by NMR spectrometer (300 Hz, Bruker, Switzerland)1H Nuclear Magnetic Resonance (NMR) spectrum. Fourier transform Infrared (FT-IR) spectra were recorded on Bruker IFS-55 (Switzerland). UV-visible spectra were recorded on a Perkinelmer Lambda 750 (Norwalk CT).
In this study, HSSG with redox response characteristics was successfully synthesized using CYS as a targeting plus anticancer linkage (fig. 1A). For comparison, non-redox reactive HCCG prodrugs were synthesized using ADH as the linkage (fig. 8).
As shown in FIG. 1B, of HA1The H NMR spectrum showed signals at 1.9 (methyl proton of N-acetyl) and 3.0-4.0 ppm (methylene and hydroxyl) and belonged to the characteristic peaks of HA. Of HA-CYS and HSSG at the same time1The H NMR spectrum showed that the peaks at 3.0-3.1 ppm were due to methylene protons of CYS, indicating that HA was linked to CYS via an amide bond between the carboxyl group of HA and the amine group of CYS. After the desired product GER-SA is successfully obtained, it is in the form of yellow oil and then used1Verification by H NMR (300 MHz, DMSO-d)6) δ 12.24 (s, 1H), 5.38-5.26 (t, J = 7.0 Hz, 1H), 5.10 (t, 1H), 4.56 (d, J = 7.0 Hz, 2H), 2.57-2.44 (m, 2H), 2.05 (m, 4H), 1.67 (s, 6H), 1.59 (d, 3H). In addition, in HSSG1In the H NMR spectrum, the olefinic proton of the GER appeared as a characteristic peak at 5.3 ppm, revealing that the GER was grafted onto the HA backbone via a redox-responsive linker (CYS). Of HA-ADH and HCCG1H NMR spectra also show similar results, demonstrating that the GER and HA are successfully linked via a non-redox-responsive linker (ADH). The amount of GER attached to the HA backbone was calculated from the integral ratio between the characteristic peak of the N-acetyl group in HA and the characteristic peak of the olefin in GER. According to1The integration values of the H NMR spectra, the degree of grafting and the Degree of Substitution (DS) of the GER in the HSSG and HCCG prodrugs were 42%/18.5% and 38%/15.4%, respectively.
In FIG. 1C, 1616 cm was observed in the FT-IR spectrum of HA-1The absorption peak at (a) is due to carbonyl stretching vibration of HA. In contrast, the absorption peak was decreased in the FT-IR spectrum of HA-CYS, which is probably due to the carboxyl group of HA. Furthermore, FT-IR spectra at 1633 and 1557 cm-1The newly generated peaks, belonging to the stretching vibration of amide I (NC = O) and the bending vibration of amide II (CN-H), are shown, indicating that an amide bond is newly formed between HA and CYS. In addition, 1540 and 1375 cm were observed in the FT-IR spectrum of HSSG-1The appearance of the peak indicates that the characteristic peak of the amide bond is changed. In addition, the characteristic peaks appeared clearly at 1670 and 2974 cm-1Due to the introduction of the GER. These similar results are also shown in the FT-IR spectra of HA-ADH and HCCG.
Characterization of HCCG NPs and HSSG NPs: the morphology was observed by a JEM 1400 JEOL Transmission Electron microscope (Tokyo, Japan) at an acceleration voltage of 200 kV.
The Drug Loading (DLC) is obtained from the following formula
Figure DEST_PATH_IMAGE003
According to1The integral of the H NMR spectrum, the degree of grafting and the Degree of Substitution (DS) of the GER in the HSSG prodrug were 42%/18.5%.
The Zeta potential, size and polydispersity index (PDI) in these micellar aqueous solutions were detected by Dynamic Light Scattering (DLS) on Malvern Zeta sizer NanoS90 (UK); and the structure and morphology of each micelle was recorded on a transmission electron microscope (TEM, JEOL JEM-1200EX microscope, Japan).
The formation of HSSG NPs is attributed to highly hydrophilic HA and poorly water soluble GER. Due to the amphiphilicity of the prodrug, HSSG prodrug conjugates can self-assemble into nanoparticles in aqueous solution. Redox sensitive nanoparticles assembled from HSSG and redox insensitive nanoparticles formed from HCCG were subjected to TEM and DLS to assess morphology and size distribution. The results confirmed the uniform spherical morphology of HSSG (fig. 2A, left) and HCCG NP (fig. 2A, right). Furthermore, according to the particle size distribution results, the HSSG and HCCG NPs have particle sizes of about 138.5 and 107.1 nm (FIG. 2B), and the polydispersity index is low (PDI < 0.200), indicating that a nanosystem with a narrow monomodal distribution is formed. The size and spherical nature may help to enhance penetration and retention as well as endocytosis. zeta potential results show that both HSSG (FIG. 10A) and HCCG NP (FIG. 10B) exhibit negative surface charges of approximately-15 mV. The negative charge is due to the HA shell, which may reduce non-specific adsorption of plasma proteins during interaction of the nanoparticles with blood components. Thus, a slightly negative zeta potential may contribute to a better stability of these nanoparticles in blood and to an increased blood circulation time.
Drug stability is an important factor in the medical application of drug delivery nanosystems. The dimensional stability of HSSG NPs was determined in PBS and PBS supplemented with 10% FBS. As shown in fig. 2C, the size of HSSG and HCCG NPs showed no significant change within 48 hours of incubation, indicating that these nanoparticles are stable enough to reach tumor sites in the blood circulation, ultimately improving drug delivery.
Example 2
The preparation method of HSSG of the present embodiment is shown in fig. 1, and comprises the following specific steps:
(1) HA (300 mg, 0.792 mmol), EDC & HCl (383 mg, 2 mmol) and NHS (230.1 mg, 2 mmol) were dissolved in 10 mL Deionized (DI) water with stirring at 25 ℃ to activate the carboxyl group of HA. After stirring for 30 min, CYS (1126.1 mg, 5 mmol) was added to the above activated NHS ester of HA and the reaction mixture was stirred for about 24 h. Thereafter, the reaction mixture was poured into a dialysis tube (MWCO 3.5kDa) and dialyzed for 72 hours. Finally, the product HA-CYS was collected by lyophilization.
(2) GER (1.54 g, 10 mmol), SA (1.50 g, 15 mmol) and DMAP (10 mg, catalytic amount) were weighed out accurately in a 200ml dry round bottom and 50ml dry dichloromethane were added. 1.582g of pyridine were added under argon and stirred at room temperature for 24 hours. After the reaction is finished, 2N hydrochloric acid and H are sequentially used2O wash and organic phase dried over magnesium sulfate. After removal of the solvent, the product GER-SA is obtained as a yellow oil.
(3) HA-CYS (476mg) was dissolved in 3 mL formamide under mild heating and cooled to 25 ℃. Then, the solution was diluted with 2mL of DMSO. GER-SA (80 mg, 0.518 mmol), EDC (80 mg, 0.521 mmol) and NHS (50 mg, 0.542 mmol) were dissolved in 5mL DMF at 25 ℃. After stirring for 30 minutes, the HA-CYS solution was introduced into the activated GER solution and then stirred at 25 ℃ for 24 hours. The final mixture was dialyzed for 72 hours (MWCO 3.5 kDa). HA-SS-GER (HSSG) was collected by lyophilization.
The comparative HA-ADH-GER (HCCG) conjugate was prepared using the same procedure as that used in example 1, except that CYS was replaced with ADH.
(4) Preparation of HCCG NPs and HSSG NPs: and (3) placing the HSSG prodrug/HCSG prodrug at 0 ℃ and 150W, performing ultrasonic treatment by a probe ultrasonic instrument, dispersing in 5mL of deionized water, and filtering the obtained ultrasonic dispersion liquid by a 0.45-micrometer membrane filter to obtain HCCG NPs and HSSG NPs.
Example 3
The preparation method of HSSG of the present embodiment is shown in fig. 1, and comprises the following specific steps:
(1) HA (300 mg, 0.792 mmol), EDC & HCl (95 mg, 0.5 mmol) and NHS (57.5 mg, 0.50 mmol) were dissolved in 10 mL Deionized (DI) water with stirring at 25 ℃ to activate the carboxyl groups of HA. After stirring for 30 min, CYS (225.2 mg, 1 mmol) was added to the above activated NHS ester of HA and the reaction mixture was stirred for about 24 h. Thereafter, the reaction mixture was poured into a dialysis tube (MWCO 3.5kDa) and dialyzed for 72 hours. Finally, the product HA-CYS was collected by lyophilization.
(2) GER (1.54 g, 10 mmol), SA (0.5 g, 5 mmol) and DMAP (10 mg, catalytic amount) were weighed out accurately in a 200ml dry round bottom and 50ml dry dichloromethane were added. 0.0791g of pyridine were added under argon and stirred at room temperature for 24 hours. After the reaction is finished, 2N hydrochloric acid and H are sequentially used2O wash and organic phase dried over magnesium sulfate. After removal of the solvent, the product GER-SA is obtained as a yellow oil.
(3) HA-CYS (476mg) was dissolved in 3 mL formamide under mild heating and cooled to 25 ℃. Then, the solution was diluted with 2mL of DMSO. GER-SA (40 mg, 0.088 mmol), EDC (40 mg, 0.261 mmol) and NHS (20 mg, 0.217 mmol) were dissolved in 5mL DMF at 25 ℃. After stirring for 30 minutes, the HA-CYS solution was introduced into the activated GER solution and then stirred at 25 ℃ for 24 hours. The final mixture was dialyzed for 72 hours (MWCO 3.5 kDa). HA-SS-GER (HSSG) was collected by lyophilization.
The comparative HA-ADH-GER (HCCG) conjugate was prepared using the same procedure as that used in example 1, except that CYS was replaced with ADH.
(4) Preparation of HCCG NPs and HSSG NPs: and (3) placing the HSSG prodrug/HCSG prodrug at 0 ℃ and 150W, performing ultrasonic treatment by a probe ultrasonic instrument, dispersing in 5mL of deionized water, and filtering the obtained ultrasonic dispersion liquid by a 0.45-micrometer membrane filter to obtain HCCG NPs and HSSG NPs.
Application example 1: multiple bioresponse drug release profiles of NPs
Specific drug release in cancer cells is crucial for the treatment of cancer by nano-therapy. It is well known that the tumor microenvironment is highly acidic, has an abnormal redox state, and contains a large amount of hyaluronidase. Thus, the multi-bioresponse drug release profile of HA-functionalized NPs was explored in various buffers.
Initially, the GER release behavior of HSSG and HCCG NPs was evaluated in buffers with different pH values (7.4, 6.5 and 5.5) used to mimic the pH values in different biological environments, including plasma (pH 7.4), tumor microenvironment (pH 6.0-6.8) and lysosomes (pH 5.5), respectively. As shown in fig. 2D, the percentage of GER release over 96 hours for HSSG and HCCG in the pH 7.4 buffer was about 20.0% and 13.8%, respectively, indicating that the HSSG and HCCG NPs retained most of the drug loading in neutral media. By lowering the pH of the buffer, the release rate of GER from NPs is significantly increased. In particular, after 96 h incubation, the cumulative percentage of GER released by HSSG and HCCG NPs into acidic buffer (pH 5.5) reached 74.7% and 69.9%, respectively, which were significantly higher than the buffer (pH 7.4). These results suggest that HSSG and HCCG NPs exhibit sustained and pH-responsive drug release patterns.
In addition, GERs are anchored to the HA chains by disulfide bonds (sensitive to GSH) and amide bonds (insensitive to GSH), which are expected to confer GSH responsibility to HSSG NPs. To verify this, we suspended the NPs in buffers (pH 6.8) containing different GSH concentrations (0.1, 1 and 10 mM) and further quantified their GER release behavior. As shown in FIG. 2E, the drug release rate of NPs was significantly accelerated with increasing GSH concentration, and about 70.2% of the drug was released cumulatively from HSSG NPs after 96 hours of incubation in buffer (pH 6.8; GSH: 10 mM), indicating that HSSG NPs showed strong GSH-responsive drug release capacity. In contrast, HCCG NPs do not exhibit this characteristic.
HA can be degraded by HAase, which is present in large numbers at the tumor site. It is therefore reasonable to speculate that NPs with HA surface functionalization could be partially destroyed due to degradation of HA, resulting in accelerated drug release. FIG. 2F shows the results of the samples containing HAase at various concentrations (0.5, 5 and 50 IUmL)-1) The release profile of GER in HSSG NPs in buffer (pH 6.8). Clearly, the rate of gastroesophageal reflux release increases with increasing concentration of HAase. In particular, in buffer (pH 6.8; HAase 50 IUmL)-1) After 96 hours of medium incubation, the cumulative GER release percentage increased from 28.5% to 86.4%.
Since multiple stimuli coexist in the tumor microenvironment, the synergistic effects of these stimuli were determined. The results show that GSH and HAase can induce the rapid GER release in 96 hours in an acidic medium. These findings clearly demonstrate a synergistic effect of stimulation on the drug release profile of HSSG NPs, which is beneficial to minimize the premature release of drug during blood circulation and to obtain sufficient intracellular drug levels.
Application example 2: in vitro cell uptake studies
1. Cell culture
LX-2 human hepatic stellate cell line and human hepatoma cell lines HepG2 and Huh7 were purchased from the cell bank of Chinese academy of sciences (Shanghai, China). All three cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% Fetal Bovine Serum (FBS), 100 μ g/ml streptomycin, and 100U/ml penicillin. Cells were incubated at 37 ℃ in 95% air and 5% CO2At 37 ℃ in a humid atmosphereAnd (5) growing.
In vitro cell uptake study
Fluorescence microscopy and flow cytometry can be used to qualitatively and quantitatively study cellular uptake, respectively. Preparation of IR 780-labeled hyaluronic acid nanoparticles: first, carboxylated IR780 is prepared from IR780 and 6-aminocaproic acid. Next, HSSG was synthesized according to the procedure in example 1. Finally, an aqueous dispersion of carboxylated IR780 and hyaluronic acid nanoparticles HSSG with free amino groups was amidated to give IR 780-labeled hyaluronic acid nanoparticles (IR 780-HSSG). 10mg of IR780 was weighed out accurately, dissolved in 5ml of methanol, and 8mg of DCC and 5mg of DMAP were added thereto, and the mixture was stirred at room temperature for 1 hour to activate the carboxyl group. After the reaction, it was purified by recrystallization. Adding a proper amount of the hyaluronic acid nano particles into HSSG aqueous dispersion, reacting for 24 hours by magnetic stirring, dialyzing (MWCO 3.5kDa) for 48 hours, removing unreacted IR780 and impurities, and freeze-drying to obtain IR 780-labeled hyaluronic acid nano particles IR 780-HSSG. The above reaction was carried out in a dark environment.
For fluorescence microscopy experiments, cells were treated with IR780-HSSG (containing 1 μ g/mL IR780) at 37 ℃ for 1,3, 6, 12 and 24 hours. Then, the cells were washed twice with PBS, fixed with 4% paraformaldehyde solution for 20 minutes, and stained with DAPI for 15 minutes. Fluorescence images were obtained using a fluorescence inverted microscope (Olympus BX43 CKX 31). The excitation wavelength of IR780 was 633 nm, and the emission spectrum was recorded between 700 and 800 nm.
The cells were treated in the same manner as in the fluorescence microscopy experiments in the flow cytometry experiments. Thereafter, the cells were washed 3 times with PBS and resuspended in 500. mu.L of PBS, and the cell uptake efficiency was examined by flow cytometry (Cyto FLEX, Beckman).
Cellular uptake is a fundamental requirement for the medical transformation of chemotherapeutic drugs in cancer therapy, since these drugs exert mainly an anticancer effect intracellularly. Thus, the cellular uptake curves of NPs were studied qualitatively and quantitatively using fluorescence microscopy and FCM, respectively.
DAPI is a blue fluorescent dye used to label the nucleus, while IR780 is a red fluorescent probe, commonly used as a fluorescent probe to track the biodistribution of nanoparticles. Therefore, the release behavior of the drug in the cell can be observed by the change of the fluorescence of IR 780. As shown in FIGS. 3A and 3B, no red fluorescence signal was observed in control cells that were not treated with IR780-HSSG NPs. In sharp contrast, cells treated with IR780-HSSG nanoparticles showed significant green fluorescence mainly in the cytoplasm after 24 hours of incubation. These observations can be attributed to the efficient cellular uptake of nanoparticles and the rapid release of GERs stimulated by HepG2 and Huh7 cellular microenvironments (acidic pH, redox state and large amount of HAase).
To further verify the cellular uptake efficiency of IR780-HSSG, quantitative analysis was performed using flow cytometry. The flow cytometer histograms in fig. 3C and 3D show that the fluorescence intensity of IR780 in Huh7 cells gradually increased and the fluorescence intensity in HepG2 cells peaked at 6h after treatment of the cells with IR780-HSSG NPs with increasing culture time (fig. 3C). These results indicate that IR780-HSSG NPs achieved high cell internalization efficiency and strong fluorescence intensity after incubation, which is very consistent with the results in fig. 3A and 3B. Therefore, HSSG NPs can be successfully internalized by HepG2 and Huh7 cells.
Application example 3: endocytic pathway assay
Culture in 6-well plates Total 3X 105HepG2 and Huh7 cells/well and incubated for 24 hours. The old medium was discarded and the prepared medium of wortmannin 10.0. mu.g/mL, genistein 50.0. mu.g/mL, CPM 10.0. mu.g/mL and HA 10mg/mL was added, respectively. After 1 hour post-pretreatment in the cell incubator, IR780-HSSG NPs solution (containing 1g/mL IR780) was added. Then, the cells were cultured continuously for 3 hours, treated, and the FCM was used to measure the fluorescence intensity.
The mechanism of enhancing drug absorption by cells by nanoparticles is an important aspect of nano-drug research. According to previous reports, the nanoparticles have two main routes to enter cancer cells, passive diffusion and active endocytosis. To better explain the mechanism of cellular uptake, the cellular uptake pathway of HSSG NPs was studied by flow cytometry. Three different endocytosis inhibitors, wortmannin, chlorpromazine hydrochloride (CPM) and genistein, were used to inhibit macropinocytosis, clathrin-mediated endocytosis and caveolin-mediated endocytosis, respectively. After addition of the different inhibitors, the mean fluorescence intensity was quantitatively measured by flow cytometry. It can be seen that the uptake of IR780-HSSG NPs by HepG2 and Huh7 cells was reduced to different extents by the addition of the three genistein, wortmannin and CPM inhibitors. Cellular uptake of HSSG by HepG2 and Huh7 cells was co-mediated by a caveolar-like invagination-mediated pathway, a macropinocytosis-mediated pathway, and a clathrin-mediated pathway. Furthermore, competitive inhibition experiments with IR780-HSSG of HA pre-incubated cells showed a significant decrease in the absorbance of HA treatment, approximately 30% and 45%, respectively. The results indicate that CD44+ specific receptor-mediated endocytosis is important for intracellular delivery of IR780-HSSG NPs.
Application example 4: in vitro cytotoxicity Studies
In order to study the inhibitory effect of the HSSG nano-system on the in vitro cancer cell activity, the MTT method was used to evaluate the cell activity of the human liver cancer cell lines HepG2 and Huh 7.
The influence of hyaluronic acid group (HA), free geraniol Group (GER), non-redox geraniol group (HCCG) and redox sensitive geraniol group (HSSG) on the survival rate of human hepatoma cell lines HepG2, Huh7 and human hepatoma cells is divided into. The evaluation of the astrocyte line LX-2 was carried out by the MTT method.
Cells in logarithmic growth phase were seeded into 96-well plates. After 24 hours of cell culture (37 ℃, 5% CO 2), 100 μ L of drugs at different concentrations were added to 96-well plates. Each concentration was set with 3 duplicate wells, set with control. After further incubation for 24, 48, 72h, the supernatant was decanted and 20. mu.L of 0.5 mg/mL MTT solution was added. After further incubation for 4h, the supernatant was discarded and 100. mu.L DMSO was added. The absorbance at 490 nm of each group was measured on a microplate reader (Multiscan, Thermo, USA). Percent survival of the treated cells was calculated and compared to untreated control cells. Cell viability (%) = OD of drug-treated cells/OD of medium-treated cells × 100%.
The toxicity of HSSG NPs on human hepatic stellate cell LX-2 cells was determined and the results in FIG. 4 indicate that these NPs are less cytotoxic to LX-2 cells than to HepG2 and Huh7 cells (0-400 μ M), even after 48 hours of co-incubation, due to the high biocompatibility of HA.
Next, the anticancer activity of HCCG and HSSG NPs against HepG2 and Huh7 cells was investigated (fig. 4A). It was found that the cytotoxicity of NPs increased with prolonged incubation time and increased drug concentration. In addition, HSSG NPs showed significant cell growth inhibition on HepG2 and Huh7 cells compared to HCCG NPs and free GERs; at the same time, the free GER was the least toxic (FIG. 4A). Thus, it can be speculated that HCCG NPs may exert cytotoxicity in the form of a prodrug, although it has no redox reaction, and thus HCCG NPs are more effective in reducing cancer cell viability than GER alone, because of the combination of CD44 receptor-mediated enhanced cellular activity absorption, drug-released cytotoxicity, and a degree of cytotoxicity without prodrug release. Meanwhile, it can be predicted that HSSG NPs will have better therapeutic effects in treating cancer. IC50 values were calculated by the software after 24h, 48h and 72h treatment of HepG2 and Huh7 cells. The results showed that the IC50 values of HCCG NPs were 90.31 and 77.58. mu.M for HepG2 and Huh7 cells, respectively, and the IC50 values of HSSG NPs were 27.24 and 27.79. mu.M, respectively, after 72 hours of exposure. It can be seen that the cell proliferation inhibitory effect of the redox-reactive HSSG NPs was about 3 times that of the non-redox HCCG NPs due to the redox-reactive drug release of the HSSG NPs. It shows that the oxidation-reduction effect of HSSG NPs is very obvious and has obvious cell killing effect.
For further study, we used concentrations of geraniol in nanoparticles of 80 and 100 μ M for HepG2 and Huh7 cells, respectively. As it proved to be approximately the median effective concentration to inhibit cell proliferation within 24 hours.
To visually assess the therapeutic effect of the drug in vitro, live/dead assays were performed by incubating HepG2 and Huh7 cells with different treatments, followed by co-staining of live and dead cells with calcein-AM (green fluorescence) and propidium iodide PI. Red fluorescence), respectively (fig. 4B). As shown in the figure. As shown in fig. 4B, HepG2 and Huh7 cells clearly observed HSSG NPs corresponding to the red fluorescence range of dead cells at GER equivalent concentrations of 80 and 100 μ M, respectively, while the red fluorescence range was much smaller for the other groups. The results are consistent with the results of the MTT measurements.
Application example 5: inhibition of proliferation and induction of cell cycle arrest in vitro
HepG2 and Huh7 cells were seeded in cell culture dishes and then incubated with HA, GER, HCCG and HSSG NPs (80. mu.M and 100. mu.M, respectively) for 24 hours. The treated cells were then stained with Calcein-AM (green, live cells) for 30 minutes and PI (red, dead cells) for 2-5 minutes. Finally, cells were imaged by fluorescence inverted microscope (Olympus BX43 CKX 31).
The 5-ethynyl-2 '-deoxyuridine (EdU) incorporation assay was performed using the Cell-Light EdU Apollo 567 in vitro imaging kit (RiboBio, Guangzhou, Guangdong, China) according to the manufacturer's instructions. HepG2 and Huh7 cells at 2X 104Individual cells/well were introduced into 96-well plates. After 24 hours of drug treatment, incubation with 10 μ M EdU for 2 hours followed by 24 hours of incubation. Then, the cells were fixed in 4% paraformaldehyde for 30 minutes, and then treated with 0.5% Triton X-100 solution at room temperature for 15 minutes. Next, 200. mu.L of Apollo solution was added to each well and incubated for 30 minutes in the absence of light. Fluorescence images were acquired using a fluorescence microscope (Olympus BX43 CKX 31). Cell proliferation rate (%) = (EdU-positive cell number)/(total cell number) × 100%.
To evaluate the role of HSSG NPs in hepatocyte proliferation, we performed EdU assay. The results show that the proliferation rates of HepG2 and Huh7 cells at 24 hours were only 5.3% and 6.4%, respectively, under the same treatment as the Calcein-AM/PI staining.
Accordingly, cell cycle analysis was subsequently performed to understand the mechanism of therapeutic effect (fig. 5H). 53. 55 for HA, GER and HCCG NPs, the proportion of cells in G2/M, G1 and S phase is similar to the cell cycle distribution of the control. In contrast, treatment of HepG2 and Huh7 cells with HSSG NPs resulted in an increase in S population and a decrease in G2/M population. Furthermore, when comparing HSSG NPs with GER and HCCG NPs, respectively, more cells arrested in S phase and fewer cells arrested in G2/M phase. Specifically, both HepG2 and Huh7 cells treated with HSSG NPs were reduced by nearly 15% after entering the S phase G2/M population compared to the control group, thereby greatly inducing cell cycle arrest. Cell cycle redistribution indicates that HSSG NPs can significantly inhibit cell proliferation.
Application example 6: in vitro apoptosis inducing effect
In situ cell death assay kit (Beyotime biotechnology, Shanghai, China) was used for terminal deoxynucleotidyl transferase mediated nicked end labeling (TUNEL) according to the manufacturer's protocol. HepG2 and Huh7 cells at 2X 104The speed of individual cells/well was transferred to 96-well plates and drug treated for 24 hours. After washing with PBS, cells were fixed in 4% paraformaldehyde for 30 minutes and then treated with 1% Triton X-100 solution at room temperature for 10 minutes. Then 200. mu.L of TUNEL solution was added to each well, incubated at 37 ℃ for 80 minutes in the dark and stained with a fluorescent dye. 4', 6-diamidino-2-phenylindole (DAPI) was used to stain nuclei for 5 min at room temperature in the dark. Fluorescence images were acquired using a fluorescence microscope (Olympus BX43 CKX 31). TUNEL positive cells were counted using Image J software.
Cells were stained with JC-1 according to the kit after various treatments as with Calcein-AM/PI staining. We then captured the fluorescence image using (Olympus BX43 CKX 31). The results are shown in FIG. 5.
Total protein was extracted from the cells. Western blots were performed to determine the expression level of the target protein. The results are shown in FIG. 5. Primary antibodies, including Anti-Bax, Anti-Bad, Anti-Bcl-xl, Anti-Bcl-2, Anti-cleared caspase-3, Anti-cleared caspase-9, Anti-cleared PARP, and Anti-GAPDH antibodies were purchased from ProteinTech (Chicago, Ill., USA). Horseradish peroxidase conjugated secondary antibodies were purchased from CST. Proteins were visualized by an enhanced chemiluminescence system (fluorochem Q, Proteinsimple, USA). Strips were semi-quantified using Image J software. The results are normalized expression levels of GAPDH.
Culture in 6-well plates Total 3X 105 HepG2And Huh7 cells/well and incubated for 24 hours. Fresh medium was then replaced and cells were treated with PBS, HA, GER, HCCG and HSSG for 24h, respectively. Apoptotic cells and cell cycles were detected by staining with YF 488-Annexin V and PI cell apoptosis detection kits (Calif., Suzhou, China) or cell cycle kits (Calif., USA) and analyzed using FlowJo software.
To further elucidate the function of HSSG NPs in inhibiting HepG2 and Huh7 cell growth, we also performed a TUNEL (TdT-mediated DUTP Nick-End Labeling) assay. As shown in fig. 5B, the HSSG NPs group had more apoptotic cells than the other groups under the same conditions. For quantitative analysis, each sample was counted and the results expressed as a percentage of TUNEL positive cells. The results showed that the apoptosis rates of the HSSG group HepG2 and Huh7 cells were 12% and 21%, respectively, which were significantly higher than those of the other groups (fig. 5D).
In addition, apoptosis was detected by flow cytometry using YF 488-annexin V/PI double staining after treatment with different nanoparticle formulations. As shown in fig. 5E, the lower left, lower right and upper right quadrants represent viable, early apoptotic and late apoptotic/necrotic regions, respectively. The results showed that there was almost no apoptosis in the control group and HA group. The apoptosis rate of HepG2 and Huh7 cells treated with GER, HCCG NPs and HSSG NPs increased in order (fig. 5G). Among all groups, HSSG NPs group showed the strongest apoptotic effect on HepG2 and Huh7 cells, resulting in apoptosis in about 14% and 41% of cells, respectively.
To reveal a potential mechanism of anti-tumor efficiency in vitro, mitochondrial membrane potential (Δ ψ m) was detected by JC-1 staining, which can lead to apoptosis when mitochondria are dysfunctional by conversion of JC-1 aggregates to JC-1 monomers in mitochondria. Mitochondria. As shown in fig. 5I, HepG2 and Huh7 cells incubated with HSSG NP had the brightest green fluorescence from JC-1 monomer staining. It was again demonstrated that HSSG NPs, which cause mitochondrial dysfunction, promote apoptosis in HepG2 and Huh7 cells more than HCCG NPs. We then tested the expression of apoptotic proteins by western blotting. Apoptosis can be regulated by a number of Bcl-2 family proteins, including anti-apoptotic proteins such as Bcl-xl and Bcl-2, and pro-apoptotic proteins such as Bad and Bax 47. Caspase is activated by a variety of apoptotic stimuli, and PARP can be further cleaved by activated Caspase-3, leading to the development of an apoptotic cascade. As shown in fig. 5J, HSSG NPs can increase the apoptosis index by up-regulating the apoptosis index of human liver cancer cells. Modulation of protein levels of cleaved cas-3, 9 and cleaved PARP indicates activation of the mitochondria-mediated apoptotic pathway.
Application example 7: in vivo biodistribution fluorescence imaging
For in vivo fluorescence imaging, H22 tumor-bearing mice were first injected with IR780-HSSG NPs by intravenous injection (IR780 concentration 10. mu.g kg-1). Fluorescence images were obtained at different post-injection times (1,3, 6, 12 and 24 hours). Subsequently, heart, liver, spleen, lung, kidney and tumor were isolated and photographed 24 hours after injection (IVIS lumine XRMS series III, usa).
To confirm the tumor internalization and accumulation capacity of IR780-HSSG NPs in vivo, H22 tumor-bearing mice were used as tumor xenograft models. Tumor accumulation and biodistribution of IR780-HSSG were monitored by the Xenogen IVIS Lumina system at predetermined time points in vivo time lapse images were obtained 0, 1,3, 6, 12 and 24 hours after IR780-HSSG NPs injection (FIG. 6E). One hour after injection, IR780-HSSG NPs began to accumulate significantly in the liver, kidney and tumor. The signal of IR780 in the tumor was highest 3 hours after injection, and slowly decreased thereafter (fig. 6E). At all time points of measurement, strong signals were found in the liver and in the tumor. This increase in liver absorption can be explained by the overexpression of the hyaluronan receptor by endocytosis on the endothelial cells of the hepatic sinus. In addition, a fluorescent signal was still observed at the tumor site 24 hours after administration, indicating that the nanoparticles have a longer retention time in vivo. The success of NPs in tumor targeting is attributed to a combination of passive Enhanced Permeation and Retention (EPR) effect and active, HA receptor-mediated cellular internalization. After 6024 hours, the mice were euthanized and tissues were removed for imaging (fig. 6F). Similar to the results of our in vivo biodistribution studies, the fluorescence signal of IR780 is mainly distributed at the tumor. At the same time, the fluorescence signals of the liver and kidney are weaker than those of tumors. This significant tumor targeting ability of HA-based nanoparticles may be due to the appropriate micelle size and hydrophilic shell of HA, which may enhance cellular uptake of NPs and promote accumulation in tumor tissues through HA-CD44 interactions. Therefore, it is expected that HSSG NPs contribute to the improvement of drug utilization efficiency and better therapeutic effect.
Application example 8: in vivo antitumor efficacy
H22 tumor-bearing mice were used as a model for assessing the anti-tumor effect of GER-loaded nanoparticles. When the volume of the transplanted tumor reaches about 150mm3At this time, the mice were randomly divided into 5 groups (6 per group) and administered at the following doses: saline group, HA group (15.5 mg/kg), free GER group (1.0mg BTZ/kg), HCCG group (1.0mg GER/kg) and HSSG group (1.0mg GER/kg). The above preparation was administered into the tail vein once every 2 days, during which time it was weighed daily, and the length (L: mm) and width (W: mm) of the tumor were measured with a vernier caliper. Tumor volume (VTumor) was calculated according to the following formula:
VTumor=W2×L/2
to evaluate the chemotherapeutic effect of HSSG NPs, H22 tumor-bearing nude mice were injected with different formulations (saline, HA, GER, HCCG NPs and HSSG NPs) via tail vein. Although the tumor growth rate (FIG. 6B) tended to increase in all groups, the growth rate of tumor size was significantly higher in HA and GER injected mice than in the group treated with HSSG NPs and HCCG NPs. This indicates that the HA-containing NPs have a targeting effect, so that the drugs are more concentrated on the tumor site, thereby obtaining a better therapeutic effect. In addition, compared with the mice treated with HCCG NPs, the mice treated with HSSG NPs had better inhibition of tumor volume growth, which indicates that after cancer cells ingest HSSG NPs, redox-reactive HSSG NPs act to cause disulfide bond cleavage and release of GER high-concentration glutathione, thereby acting as chemotherapy. Mice injected with saline and HA showed the fastest tumor growth. Meanwhile, optical photographs of the tumors (fig. 6A) and tumor weights (fig. 6C) at 14 days post-injection also demonstrate that the tumors of HSSG NPs treated mice had the least weight and the best therapeutic effect.
To further confirm the anti-tumor effect in vivo, tumors were excised and tested for HE staining, Ki-67 and TUNEL. As shown in fig. 6D, the control group showed typical characteristics of tumor tissue in HE staining, such as large and deep nuclei and less cytoplasm. In contrast, the tumor tissues of the treated groups showed different degrees of necrosis, such as nuclear shrinkage and increased cytoplasmic area. The HSSG NPs group showed the largest tumor necrosis zone. FIG. 6D shows images of immunohistochemical staining with Ki-67. The results showed that the most Ki-67 positive cells were in the saline-treated group. However, the number of Ki-67 positive cells decreased after treatment with HSSG NPs. In addition, groups of tumor cells were analyzed for apoptosis by TUNEL sections (fig. 6D). DAPI stained the nuclei blue, and the nuclear DNA of tumor cells was destroyed during early apoptosis and marked green by FITC. Large areas of green fluorescence were found in HSSG NPs, indicating that the drug induced apoptosis of tumor cells. These data indicate that inhibition of cell proliferation and promotion of apoptosis by HSSG NPs are better than other drugs.
Application example 9: systemic toxicity assessment
Blood samples of healthy mice were collected in containers containing heparin and centrifuged at 1500 rpm for 10 minutes to obtain Red Blood Cells (RBCs). After washing and dilution with PBS, RBCs were mixed with HSSG and HCCG NPs, dispersed in PBS at various concentrations, and incubated at 37 ℃. After 3 hours of incubation, the mixed solution was centrifuged and centrifuged at 570 nm (A)sample) The absorbance of the supernatant was measured. At the same time, erythrocytes were mixed with PBS and water as negative control (APBS) and positive control (A), respectivelywater). Percent hemolysis is defined by (A)sample-APBS)/(Awater-APBS) X 100% calculation.
After 14 days of treatment, all mice were sacrificed and tumors were collected and weighed. The major organs such as heart, liver, spleen, lung, kidney, and tumor were carefully excised, stored with 4.0% (w/v) paraformaldehyde, embedded in paraffin, and further processed into immunohistochemical sections. The samples were cut into 7 μm thick sections and stained with hematoxylin and eosin. Tumor tissue was cut into 5 μm thick sections and stained with rabbit polyclonal CD31 antibody (1: 200) and rabbit polyclonal anti-Ki-67 antibody (1: 500). In addition, TUNEL (transferase mediated dUTP-biotin nick end labeling) and immunofluorescence labeling (FITC labeling) were used to study the apoptosis of tumor cells, and tumor cells of different formulation groups were compared for apoptosis, and the treatment effect was evaluated and analyzed. Each group of effects. The slides were observed using a fluorescence microscope.
Hemolysis assay was performed to study HSSG and HCCG NP in blood (fig. 9). No visible haemolysis was observed within the range tested. Functional markers, including Creatinine (CREA), urea, aspartate Aminotransferase (AST), and alanine Aminotransferase (ALT), are detected biochemically in the blood. CREA, urea, AST and ALT levels on day 14 were in different groups (fig. 7B and 7C). In addition, no significant abnormalities were found in body weight after the different treatments (fig. 7A). HE staining of the body weight, major organs (heart, liver, spleen, lung and kidney) and blood biochemical results indicate that HSSG NPs treatment is safe and biocompatible. In addition, hematoxylin of major organs (heart, liver, spleen, lung and kidney). Eosin (HE) staining showed no apparent damage in all groups (fig. 7D).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (10)

1. A hyaluronic acid-geraniol polymer prodrug multi-bioresponse drug delivery system HSSG NPs having the structure:
Figure DEST_PATH_IMAGE001
。
2. the delivery system HSSG NPs of claim 1, wherein: the drug delivery system HSSG NPs are synthesized by self-assembling NPs through disulfide bonding of hydrophilic groups HA and hydrophobic groups GER on HSSG.
3. The delivery system HSSG NPs of claim 1, wherein: the delivery system HSSG NPs have pH/glutathione/hyaluronidase responsive drug release capacity.
4. The method for preparing the delivery system HSSG NPs of any of claims 1-3, characterized by the following steps:
(1) dissolving HA, EDC & HCl and NHS in deionized water, adding CYS after stirring, reacting completely under stirring, dialyzing the reaction mixture, and freeze-drying to obtain HA-CYS;
(2) adding anhydrous dichloromethane into a mixture of GER, SA and DMAP, adding pyridine under the protection of argon, completely reacting under the condition of stirring, washing and drying the reaction mixture, and removing a solvent to obtain GER-SA;
(3) dissolving the HA-CYS obtained in the step (1) in formamide, cooling, and then diluting with DMSO to obtain an HA-CYS solution; dissolving GER-SA, EDC and NHS obtained in the step (2) in DMF, and stirring and activating to obtain a GER solution; then adding the HA-CYS solution into the GER solution, reacting completely under the condition of stirring, dialyzing and freeze-drying the obtained mixture to obtain HA-SS-GER (HSSG);
(4) and (4) dispersing the HSSG obtained in the step (3) in deionized water after ultrasonic treatment, and filtering the obtained ultrasonic dispersion liquid through a membrane filter to obtain HSSG NPs.
5. The method of claim 4, wherein: the molar ratio of HA, EDC & HCl, NHS and CYS in the step (1) is 0.792, (0.5-2), (1-5).
6. The method of claim 4, wherein: the molar ratio of GER, SA and pyridine in the step (2) is 10 (5-15) to 0.001-0.02; DMAP was added in an amount of 1mg DMAP per mmol of GER.
7. The method of claim 4, wherein: the mass ratio of HA-CYS, GER-SA, EDC and NHS in the step (3) is 476 (40-80): (40-80): (20-50).
8. The method of claim 4, wherein: the temperature of ultrasonic treatment in the step (4) is 0 ℃, and the power is 150W; the diameter of the membrane filter was 0.45 μm.
9. Use of the delivery system HSSG NPs according to claim 1 or 2 for the manufacture of a medicament targeting CD44 receptor.
10. Use of the delivery system HSSG NPs according to claim 1 or 2 for the preparation of an anti-tumor medicament, characterized in that: the application size of the drug delivery system HSSG NPs is 101.7nm, and the drug loading efficiency is 18.5%.
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