CN117771185A - Layered targeting dual delivery system based on photo-immunotherapy and preparation method and application thereof - Google Patents
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- CN117771185A CN117771185A CN202311835382.5A CN202311835382A CN117771185A CN 117771185 A CN117771185 A CN 117771185A CN 202311835382 A CN202311835382 A CN 202311835382A CN 117771185 A CN117771185 A CN 117771185A
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Abstract
The invention discloses a layered targeting dual delivery system based on photo-immunotherapy and a preparation method and application thereof. The layered targeting dual delivery system is mainly formed by preparing a compound from a photosensitizer, a phenylboronic acid derivative and hyaluronic acid, then inserting the compound into a phospholipid membrane formed by phospholipid and cholesterol, and finally encapsulating an IDO inhibitor. The delivery system is capable of aggregating at the tumor site through long circulation and EPR effects. Subsequently, the phenylboronic acid ester bond is destroyed by the weakly acidic environment of the tumor tissue, exposing phenylboronic acid groups that are specifically recognized by sialic acid residues, thereby enhancing the specific uptake by tumor cells. The layered targeting dual delivery system based on the photo-immunotherapy designed by the invention can treat melanoma, and can better play the role of inhibiting proliferation and metastasis of the melanoma by the photo-immunotherapy.
Description
Technical Field
The invention belongs to the technical field of drug targeted delivery systems, and particularly relates to a layered targeted dual delivery system based on photo-immunotherapy, and a preparation method and application thereof.
Background
Melanoma is a malignant skin cancer that originates in melanocytes and has a very high tendency to metastasize and is fatal. In the early stages, most melanoma patients can be cured by surgical excision. However, once metastasis occurs in melanoma, it is difficult to effectively cure melanoma. Thus, developing an effective combination therapy regimen and intelligent nanodelivery system is of great importance to increase immune response rates, selectively destroy solid tumors, and eliminate metastatic lesions.
Phototherapy is an anti-tumor therapy combining phototherapy with immunotherapy, which can effectively enhance immune stimulation response while eliminating solid tumors, thereby exerting synergistic anti-tumor metastasis effect. Phototherapy includes photothermal therapy (PTT) and photodynamic therapy (PDT). Under the irradiation of light with a certain wavelength, PTT generates high heat through non-radiative decay, so that the local temperature of cells and tissues is increased, and tumor ablation is realized. While PDT kills tumor cells by generating Reactive Oxygen Species (ROS) to trigger cytotoxicity. Phototherapy was found to not only destroy tumor cells, but also induce Immunogenic Cell Death (ICD). In particular, local hyperthermia or ROS injury induces the release of Tumor Associated Antigens (TAAs) and injury associated molecular patterns (DAMPs), the release of DAMPs including the secretion of Adenosine Triphosphate (ATP), the exposure of Calreticulin (CRT) and the release of high mobility histone B1 (HMGB 1). ATP serves as a chemoattractant to recruit Antigen Presenting Cells (APCs). CRT serves as a "eat me" signal that promotes APC to destroy dying tumor cells and their fragments. HMGB1 stimulates antigen presentation to T cells. As immune activators, released TAAs and DAMPs can stimulate maturation of Dendritic Cells (DCs). Mature DCs carry antigen to the lymph nodes and activate effector T cells. Finally, activated effector T cells migrate and infiltrate tumor tissue, thereby achieving the goal of destroying tumor cells.
Rhodopsin 18 is a dihydroporphyrin derivative of chlorophyll and can be used as a photosensitizer for PDT and PTT. The rhodopsin 18 mediated phototherapy induced immune response is stronger compared to single PDT or PTT. However, phototherapy-induced immune responses are insufficient to eliminate the proliferation and metastasis risk of melanoma. The tumor cells can also be camouflaged by utilizing various immune escape mechanisms in the tumor microenvironment, so that the exertion of the anti-tumor curative effect is severely limited. Based on this, a combined administration scheme is developed, and the inhibition effect of the photo-immunotherapy on the proliferation and metastasis of melanoma is expected to be better exerted by inhibiting the immune escape mechanism of tumor cells and enhancing the immune response level at the same time of the photo-therapy.
Currently, modulation of immune responses by altering the metabolism of immune cells is an attractive strategy. Indoleamine-2, 3 dioxygenase (IDO) is an enzyme that catalyzes the degradation of tryptophan (Trp) to kynurenine (Kyn), playing an important role in the metabolic process of immune cells. IDO has been shown to be one of the murders of immunosuppressive microenvironments, and Trp depletion accelerates the growth of regulatory T cells (Tregs) while inhibiting the activation of cytotoxic T Cells (CTLs). Clinical researches show that IDO inhibitors such as indoximod, NLG919 and Epacadostat (EPA) can effectively inhibit IDO activity and inhibit immune escape of tumor cells, thereby improving anti-tumor treatment effect. Therefore, the combined administration scheme of the photosensitizer and the IDO inhibitor is hopeful to enhance the immune response effect of the organism and realize high-efficiency anti-tumor.
Targeting nanocarriers are considered ideal carriers for delivering photosensitizers and IDO inhibitors to enhance tumor penetration and cellular internalization. Hydrophilic material modified nano-drug carrier (100-200 nm) has long circulation characteristic, and is easier to accumulate at tumor sites through EPR effect. Hyaluronic acid is a hydrophilic natural linear disaccharide polymer, can improve tumor targeting and biocompatibility of the nanoparticles, and prolongs the circulation time of the nanoparticles in vivo. In the tumor microenvironment, the CD44 receptor on the surface of tumor cells can be specifically targeted. However, hyaluronic acid is easily and rapidly degraded in vivo, resulting in insufficient ability of the nano-formulation to target cell internalization. In addition, some small molecule targets are readily recognized specifically by melanoma cells. For example, sialic acid residues are over-expressed on the cell membrane due to aberrant glycosylation and are a sign of poor prognosis in melanoma patients. Sialic acid residues can be targeted by antibodies, lectins or phenylboronic acids. In particular, phenylboronic acid shows good physiological stability under the acidic pH condition (pH 6.5) of tumors, and has higher selectivity and binding affinity for sialic acid residues. However, some normal tissues, especially liver and lung tissues, also express sialic acid residues to some extent, which inevitably results in off-target effects.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention designs a layered targeting dual delivery system based on the photo-immunotherapy for treating melanoma, which better plays the role of inhibiting proliferation and metastasis of the melanoma by the photo-immunotherapy.
The technical scheme is as follows: in order to solve the problems, the invention adopts the following technical scheme:
a layered targeting dual delivery system based on photo-immunotherapy is mainly formed by preparing a complex from a photosensitizer, a phenylboronic acid derivative and hyaluronic acid, inserting the complex into a phospholipid membrane formed by phospholipid and cholesterol, and finally encapsulating an IDO inhibitor.
This hierarchical targeting strategy is implemented in two stages: (1) Modification of hydrophilic materials (hyaluronic acid) can prolong circulation time in vivo, and then target tumor tissues through EPR effect; (2) Targeting tumor cells based on small molecule targeting ligands such as phenylboronic acid.
Preferably, the photosensitizer is selected from the group consisting of rhodopsin 18; the IDO inhibitor is selected from one of indoximod, NLG919 and Epacadostat (EPA).
Preferably, the phenylboronic acid derivative is selected from 4-aminomethylphenylborates; the molecular weight of the hyaluronic acid is 5000Da-10000Da; the phospholipid is one of natural phospholipid, soybean lecithin and egg yolk lecithin.
The invention also provides a preparation method of the layered targeting dual delivery system based on the photo-immunotherapy, which comprises the following steps:
(1) Dissolving a photosensitizer and a condensing agent, activating, adding phenylboronic acid derivatives in a stirring state, and stirring at room temperature for reaction to obtain a photosensitizer-phenylboronic acid compound;
(2) Respectively dissolving hyaluronic acid and photosensitizer-phenylboronic acid compound, dropwise adding photosensitizer-phenylboronic acid compound solution into hyaluronic acid solution under stirring, adjusting pH, and stirring at a certain temperature for reaction to obtain photosensitizer-phenylboronic acid-hyaluronic acid compound;
(3) Taking phospholipid and cholesterol, preparing a lipid film, adding a solution containing a photosensitizer-phenylboronic acid-hyaluronic acid compound for hydration, and performing ultrasonic treatment to obtain a compound-liposome solution;
(4) And (3) dropwise adding the solution containing the IDO inhibitor into the complex-liposome solution under the stirring condition, incubating, and removing unencapsulated IDO inhibitor to obtain the layered targeted dual delivery system.
Preferably, in the step (1), the condensing agent is selected from one or more of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4-Dimethylaminopyridine (DMAP) and 1-Hydroxybenzotriazole (HOBT); the mol ratio of the photosensitizer to the phenylboronic acid derivative is 1 (2-4); the reaction time is 22-26h.
Preferably, in the step (2), the molar ratio of the photosensitizer-phenylboronic acid complex to the hyaluronic acid is (4-6): 1; the pH is adjusted to 8-10; the reaction temperature is 60-70 ℃, and the reaction time is 22-26h.
Preferably, in the step (3), the lipid film is prepared by a film dispersion method; the mass ratio of the phospholipid to the cholesterol is (8-10): 1; the mass ratio of the phospholipid to the photosensitizer-phenylboronic acid-hyaluronic acid complex is (3-5) 1; the solution containing photosensitizer-phenylboronic acid-hyaluronic acid complex is prepared by adopting (NH) 4 ) 2 SO 4 Solution of (NH) 4 ) 2 SO 4 The concentration of the solution is 200-300mM; the hydration temperature is 30-50 ℃, and the hydration time is 50-70min; the ultrasonic treatment adopts probe ultrasonic treatment, the ultrasonic power is 250-350W, and the ultrasonic treatment time is 4-6min; removing (NH) by dialysis after ultrasonic treatment 4 ) 2 SO 4 。
Preferably, in the step (4), the mass ratio of the IDO inhibitor to the photosensitizer is (4-6) 1; the incubation temperature is 30-50 ℃, and the incubation time is 20-40min.
The invention finally provides application of the layering targeting dual delivery system based on the photo-immunotherapy in preparation of drugs for treating melanoma.
Further, the drug for treating melanoma can inhibit proliferation and metastasis of melanoma.
The layered targeting dual delivery system (lip\EPA\P 18-APBA-HA) constructed by the invention HAs the advantages of simple preparation method, higher drug loading capacity, about 180nm particle size, good photo-thermal trigger drug release performance and serum stability. In lip\epa\p18-APBA-HA, the hyaluronic acid coating is to extend circulation time in vivo and target tumor tissue by EPR effect. Subsequently, the phenylboronic acid ester bond with pH sensitivity is destroyed by the weak acidic microenvironment of tumor tissues, exposing phenylboronic acid groups which can be specifically recognized by sialic acid residues on the surface of tumor cells, and showing the potential of targeting tumor cells. This layered targeting strategy facilitates drug distribution in tumor tissues and receptor-mediated efficient cellular uptake. The lip\EPA\P18-APBA-HA is more easily internalized by tumor cells through a layered targeting design, and the release of EPA is triggered under the action of light and heat, so that the activity of IDO is inhibited, and the activation of the immune response of the organism is promoted. Therefore, lip\EPA\P18-APBA-HA can induce anti-tumor immunity by more effectively promoting DCs maturation, CTLs cell activation, inhibiting Tregs and regulating cytokine secretion, thereby effectively inhibiting proliferation and metastasis of melanoma. In summary, the combination of the photosensitizer and the IDO inhibitor provided by the invention is a promising photo-immunotherapy strategy for treating melanoma, and the layered targeting nano-delivery system is a new hope for better exerting the inhibition effect of photo-immunotherapy on proliferation and metastasis of melanoma.
In conclusion, the photosensitizer is connected with the phenylboronic acid derivative through an amide bond, and then the hyaluronic acid is connected through a phenylboronic acid ester bond. Hyaluronic acid can cover phenylboronic acid groups and target CD44 receptors of tumor tissues, the phenylboronic acid groups are positioned to the tumor tissues, then the phenylboronic acid groups which can be combined with sialic acid residues are exposed after the hyaluronic acid is removed through lower pH of the tumor microenvironment, so that the problem of off-target is solved; and as the compound prepared from the hyaluronic acid, the phenylboronic acid derivative and the photosensitizer is inserted into the phospholipid membrane, the stability of the carrier can be obviously improved through the hydrophilic shielding effect of the surface hyaluronic acid, and finally the targeting effect of the carrier is further improved.
The beneficial effects are that: the invention creatively provides a layering targeting dual delivery system based on photo-immunotherapy, which has uniform particle size and good photo-thermal trigger drug release performance and serum stability. The layered targeting dual delivery system realizes the efficient co-delivery of the photosensitizer and the IDO inhibitor and the synergistic induction of the anti-tumor immunity of organisms, shows extremely strong capacity of inhibiting the proliferation and the metastasis of melanoma, and has potential clinical application value.
Drawings
FIG. 1 is a schematic diagram of P18-APBA-HA according to the present invention 1 H-NMR spectrum.
FIG. 2 is a graph showing the results of preparation and characterization of lip\EPA\P18-APBA-HA according to the present invention.
FIG. 3 is a graph of the results of in vitro photothermal investigation of lip\EPA\P18-APBA-HA of the present invention.
FIG. 4 is a graph of the results of pH triggered lip\EPA\P18-APBA-HA particle size and zeta potential changes of the present invention.
FIG. 5 is a graph showing the serum stability and in vitro drug release results of lip\EPA\P18-APBA-HA of the present invention.
FIG. 6 is a graph showing the results of in vitro cell uptake experiments of lip\EPA\P18-APBA-HA of the present invention.
FIG. 7 is a graph of the results of in vitro ROS production experiments of lip\EPA\P18-APBA-HA of the present invention.
FIG. 8 is a graph showing the results of in vitro cytotoxicity studies of lip\EPA\P18-APBA-HA according to the present invention.
FIG. 9 is a graph showing the results of in vitro ICD induction experiments with lip\EPA\P18-APBA-HA of the present invention.
FIG. 10 is a graph showing the tissue distribution of lip\EPA\P18-APBA-HA according to the present invention.
FIG. 11 is a graph showing the results of in vivo anti-tumor efficacy of lip\EPA\P18-APBA-HA according to the present invention.
FIG. 12 is a graph showing the results of experiments for in vivo maturation of induced DCs in Lip\EPA\P18-APBA-HA according to the present invention.
FIG. 13 is a graph showing the results of in vivo IDO inhibition experiments with lip\EPA\P18-APBA-HA according to the present invention.
FIG. 14 is a graph showing the results of in vivo promotion of immune-related cytokine secretion by lip\EPA\P18-APBA-HA of the present invention.
FIG. 15 is a graph showing the results of in vivo activation of Ths, CTLs and inhibition of Tregs by lip\EPA\P18-APBA-HA of the present invention.
FIG. 16 is a graph showing the results of in vivo anti-tumor pulmonary metastasis therapy with lip\EPA\P18-APBA-HA of the present invention.
Detailed Description
The invention will be better understood from the following examples. However, the description of the embodiments is only for illustrating the invention and should not limit the invention described in detail in the claims.
EXAMPLE 1 Synthesis and characterization of the P18-APBA-HA Structure
The experimental method comprises the following steps: 50mg of rhodopsin 18 (P18), 18.3mg of EDCI and 11.5mg of DMAP are dissolved in 2mL of DMSO and stirred at room temperatureActivated by stirring, 56.4mg of 4-Aminomethyl Phenylborate (APBA) was added thereto while stirring, and the mixture was stirred at room temperature for 24 hours. Dialyzing the reaction mixture with water for 24 hr, lyophilizing to obtain red-purple solid photosensitizer-phenylboronic acid complex (P18-APBA), and making the obtained product pass through 1 H-NMR was used for characterization.
40mg of Hyaluronic Acid (HA) having a molecular weight of 8000Da was dissolved in 2mL of formamide, and 14mg of P18-APBA was dissolved in 2mL of DMSO. The P18-APBA solution was then added to the HA solution with stirring. Subsequently, caH is added 2 Adjusting pH to=9, stirring at 65deg.C for 24 hr, dialyzing the reaction mixture with water overnight, and lyophilizing to obtain olive green solid photosensitizer-phenylboronic acid-hyaluronic acid complex (P18-APBA-HA), and making the obtained product pass through 1 H-NMR was used for characterization.
Experimental results: P18-APBA-HA 1 The H-NMR spectrum is shown in FIG. 1, and proton signal peaks of 6.5 to 8.0 and 9.25ppm are assigned to functional APBA fragment, while other proton signals are assigned to P18 fragment. The amide proton signal showed a peak at 9.25ppm indicating that APBA was linked to P18. APBA-P18 can be covalently bound to the diol group of HA, the structure of P18-APBA-HA being 1 H-NMR confirmed. The characteristic peak of HA appears at 1.82ppm (N-acetylmethyl proton, -NHCOCH 3 ) And 3.30 to 4.58ppm (hydroxyl groups and methylene groups of the polysaccharide backbone). The methyl proton peak of P18 appears at 1.02-1.30 ppm, and the characteristic peak of phenyl appears at 7.92-8.31 ppm, indicating successful synthesis of P18-APBA-HA.
Example 2 preparation and characterization of lip EPA P18 APBA HA
The experimental method comprises the following steps: 18mg of soybean lecithin and 2mg of cholesterol were placed in a round bottom flask, 2mL of chloroform was added for dissolution, and the solvent was removed by a rotary evaporator to form a lipid film. Then, the round bottom flask was placed in a 40℃water bath, to which 4mL 250mM (NH) 4 ) 2 SO 4 The solution was hydrated for 1h (containing 5mg of P18-APBA-HA). The complex liposome solution (lip\P18-APBA-HA) was obtained by probe ultrasound (300W, 5 min). Removing (NH) outside Lip\P18-APBA-HA by dialysis with PBS solution of 0.01M and pH 7.4 as dialysis medium 4 ) 2 SO 4 . Subsequently, lip\P18-APBA-HA was transferred to a 40℃water bath and EPA was dissolvedThe solution was added dropwise to lip\P18-APBA-HA under stirring and incubated for 30min. Collecting the solution, and dialyzing with PBS solution of 0.01M and pH 7.4 as dialysis medium to remove non-encapsulated medicine to obtain lip\EPA\P18-APBA-HA. The particle size, PDI and zeta potential were measured using a nanoparticle potentiometer. The liposomes were morphologically observed by negative staining. Briefly, liposomes were adsorbed onto copper grids and stained with 1% phosphotungstic acid solution. The morphology was then observed with a Transmission Electron Microscope (TEM) at an accelerating voltage of 80 kV.
And respectively adopting a high performance liquid chromatography method and an ultraviolet spectrophotometry method to measure the drug contents of EPA and P18 in the liposome. In the detection of EPA, a certain amount of drug-loaded liposome was dissolved in acetonitrile, EPA was extracted by ultrasonic treatment for 20min, and after filtration through a 0.45 μm membrane, the EPA content was measured at 290nm using acetonitrile/water (70:30, v/v) as a mobile phase. The mobile phase was passed through an ODS-3C18 column at a rate of 1 mL/min. For the detection of P18, the OD value was determined at 406nm after treatment with methanol in the same manner. Finally, the drug Loading (LC) and encapsulation (LE) rates of EPA and P18 were calculated.
Experimental results: as shown in FIG. 2, the research synthesizes P18-APBA-HA with phenylboronic acid ester bond for the first time, and uses the P18-APBA-HA as a liposome modification material to construct a HA modified layered targeting dual delivery system (lip\EPA\P 18-APBA-HA). The particle size of lip\EPA\P18-APBA-HA was slightly larger than that of the blank or unmodified liposome, and was about 180nm. The particle size distribution range of the liposome is 143-180 nm, and is consistent with the passive targeting particle size range of 100-200 nm. All liposomes had a polydispersity index (PDI) of less than 0.3, indicating stable formulation and a smaller particle size distribution of the liposomes produced (fig. 2A). The drug-loaded liposomes are negatively charged. Wherein lip\EPA\P18-APBA-HA HAs the lowest surface zeta potential, which may be due to surface modification of P18-APBA-HA (FIG. 2B). The P18-APBA-HA and EPA are co-loaded in a liposome system by adopting a drug loading method combining a passive method and an active method. As shown in fig. 2C and 2D, all liposomes had an encapsulation efficiency of greater than 70% for EPA or P18, with ideal loading capacity. TEM results show that lip\EPA\P18-APBA-HA is nearly circular, HAs a fingerprint structure at the edge, and is blurred, probably due to the surface modification of HA. Meanwhile, structures of lip\EPA\P18 and lip\EPA\P18-APBA are observed through TEM. Both are nearly circular, with a clearer fingerprint structure (fig. 2E).
Example 3 in vitro photothermal investigation of lip/EPA/P18-APBA-HA
The experimental method comprises the following steps: in this experiment, PBS was used as a control, and the photo-thermal properties of the P18 solution were evaluated by detecting the temperature change of the solution under 671nm laser irradiation. Preparation of P18 solutions of different concentrations at 0.8W/cm 2 The optimum concentration of the P18 solution was selected by irradiating the 671nm laser light of (1) for 5min, and the optimum concentration and power of the P18 photothermal conversion were determined by irradiating the 671nm laser light of different powers for 5 min. In the experiment, PBS is used as a control, and in-vitro photo-thermal properties of EPA+P18, lip\EPA\ P18, lip\EPA\P18-APBA and lip\EPA\P18-APBA-HA are detected by the same method.
Furthermore, to evaluate the photo-thermal stability of lip\epa\p18-APBA-HA, we irradiated lip\epa\p18-APBA-HA for 5min at the same conditions with optimal concentration and power, then shut down the laser for 5min for cooling, and the cycle was repeated 4 times. The temperature was recorded every 30s using an infrared thermal imaging system. The calculation formula for Δt is as follows: Δt=t s -T 0 (T s And T 0 Representing the sample temperature and the starting temperature, respectively).
Experimental results: results as shown in fig. 3, to evaluate the photo-thermal properties of P18, we monitored the in vitro photo-thermal conversion properties of P18 at different concentrations and different powers using a thermal infrared imager (fig. 3A and B). First, at 0.8W/cm 2 Under power conditions, the effect of P18 concentration on the photothermal conversion efficiency was studied. At 0.8W/cm 2 After 5min of laser irradiation, the temperature of the P18 solution rapidly increased, and the temperature amplitude increased with the increase of the P18 concentration. At a P18 concentration of 2. Mu.g/mL, the temperature reached the temperature range of light PTT (. Gtoreq.42 ℃) after 5min of laser irradiation. When the concentration of P18 was 5. Mu.g/mL, the temperature reached 44℃after 5min of laser irradiation. Literature shows that high temperature PTT>43℃) can cause irreversible damage to tumor cells. We continue to study the effect of laser power on the efficiency of photothermal conversion at a relatively medium concentration of 2.5 μg/mL P18. The result shows that the laser power has a larger influence on the photo-thermal performance of P18. When the power exceeds 0.8W/cm 2 At this time, the temperature rise of the P18 solution was large within 5 min. Subsequently, we continued to investigate EPA+P18, lip\EPA\P18-APBA and lip\EPA\P18-APBA-HA at a concentration of 2.5. Mu.g/mL at 0.8W/cm 2 Photo-thermal properties under laser irradiation. As shown in fig. 3C and D, fluke PTi120 thermal imagers recorded infrared thermal images and temperature changes at different times. P18-APBA-HA, lip\EPA\P18-APBA and lip\EPA\P18-APBA-HA have photo-thermal characteristics as P18. This suggests that P18 derived materials can retain the potential of PTT by direct insertion into liposomes. To investigate the photo-thermal stability of Lip\EPA\P18-APBA-HA (2.5. Mu.g/mL P18), it was exposed to 0.8W/cm 2 Is continuously turned on/off (5/5 min) for 4 cycles under 671nm laser (FIG. 3E). In 4 cycles, the thermal fatigue performance loss is very small, which indicates that the lip\EPA\P18-APBA-HA HAs strong photo-thermal stability.
EXAMPLE 4pH triggered Lip\EPA\P18-APBA-HA particle size and zeta potential variation
The experimental method comprises the following steps: lip\EPA\P18-APBA-HA was added to PBS (0.01M) at pH 6.5 or pH 7.4, respectively, incubated at 37℃and the diameter and zeta potential of the Lip\EPA\P18-APBA-HA were measured at predetermined time points.
Experimental results: particle size and zeta potential changes of lip\EPA\P18-APBA-HA under different pH conditions are shown in FIG. 4. At pH 7.4, the particle size and zeta potential of the Lip\EPA\P18-APBA-HA changed little. However, at pH 6.5, the particle size of Lip\EPA\P18-APBA-HA was reduced by about 25nm and the zeta potential was increased by-12 mV. It is speculated that the changes in particle size and zeta potential of lip\EPA\P18-APBA-HA may be due to the removal of the externally modified HA by phenylboronic acid bond cleavage in an environment of pH 6.5.
EXAMPLE 5 serum stability and in vitro drug Release investigation of lip/EPA/P18-APBA-HA
The experimental method comprises the following steps: the stability of liposomes to serum reactions was assessed by measuring particle size changes. Lip\EPA\P18, lip\EPA\P18-APBA and lip\EPA\P18-APBA-HA were added to pH 7.4PBS (0.01M) containing 10% (v/v) fetal bovine serum, incubated at 37℃for 24 hours, and the particle size of the liposomes was measured by a nanoparticle potentiometer at predetermined time points.
Evaluation of Li by dialysisThe release behavior of EPA in p\EPA\P18-APBA-HA was mediated by PBS (0.01M, 0.25% Tween-80) at pH 5.5, 6.5 and 7.4, respectively. Exposure to 6711 nm 0.8W/cm before dialysis 2 Liposome groups at 5min under laser are denoted by L+. Lip\EPA\P18-APBA-HA was placed in a dialysis bag (MWCO, 3.5 kDa), dispersed in 20mL of release medium, and placed in a thermostatted shaker (100 rpm,37 ℃). 0.5mL of the solution was collected at the predetermined time point and replenished with the same amount of fresh medium, and finally the cumulative release of EPA was determined by high performance liquid chromatography.
Experimental results: as shown in FIG. 5A, the particle size change of lip\EPA\P18-APBA-HA was kept around 10nm, indicating that lip\EPA\P18-APBA-HA had good serum stability. However, the particle size of the lip\EPA\P18 and lip\EPA\P18-APBA groups increased rapidly after 1h of incubation and then remained stable. The results show that the hydrophilic modification of HA is more beneficial to improving the stability of the liposome in serum and the circulation time of the liposome in vivo. During the culture process, the lip\EPA\P18 and lip\EPA\P18-APBA groups may bind to serum proteins, thereby rapidly increasing the diameter.
We also studied the drug release behavior of lip\EPA\P18-APBA-HA at different pH values (FIG. 5B). The results indicate that pH affects the drug release behavior of EPA in lip\EPA\P18-APBA-HA, probably due to the removal of HA at lower pH, thus reducing the stability of the liposome system. In addition, the release rate of EPA in lip\EPA\P18-APBA-HA was significantly increased after laser irradiation. This is probably because P18-APBA-HA surface-modifies liposomes mainly by inserting hydrophobic moieties (P18) into the phospholipid bilayer. Upon laser irradiation, P18-APBA-HA mediates a temperature rise, which changes the fluidity of the phospholipid bilayer, thereby facilitating EPA release from the liposome.
Example 6 in vitro cell uptake experiment of lip/EPA/P18-APBA-HA
The experimental method comprises the following steps: B16F10 cells (6X 10) 3 Individual/well) was inoculated into a ViewPlate-96 microplate, cultured overnight, and then treated with Rhodamine B (RB) -labeled liposomes Lip\RB\P18, lip\RB\P18-APBA-HA and Free RB (RB 10. Mu.g/mL, P18.25. Mu.g/mL), respectivelyAnd (5) treating for 2 or 4 hours. For competition experiments, cells were pre-incubated with HA or APBA for 30min, followed by Lip\RB\P18-APBA-HA. After the incubation, the cells were washed with PBS, fixed with immunostaining fixative at room temperature for 10min, and then stained with Hoechst 33342. Finally, the liposomes were captured for cellular uptake using an operatta CLS high content imaging system.
Experimental results: as shown in FIG. 6, at 2 and 4 hours, the fluorescence signal of Free RB in tumor cells was weak, while the fluorescence intensity of RB-labeled liposomes was significantly enhanced. This is probably due to passive diffusion of Free RB in cells, whereas liposomes can enter cells through fusion and endocytosis of the liposomes with cell membranes, thus exhibiting higher cellular uptake efficiency. Under the culture condition of a culture medium with the pH of 6.5, the uptake of the tumor cells to the lip\RB\P18-APBA and the lip\RB\P18-APBA-HA is observed to be obviously superior to that of the lip\RB\P18 group. To investigate whether the enhancement of lip\rb\p18-APBA-HA uptake was mediated through the HA-CD44 ligand pathway, we pre-incubated HA in advance to saturate the CD44 receptor on the B16F10 cell surface. We observed that after blocking CD44 receptor, B16F10 uptake of lip\rb\p18-APBA-HA was not significantly reduced, suggesting that uptake of lip\rb\p18-APBA-HA by B16F10 cells may be primarily dependent on other pathways. Therefore, we preincubated APBA in advance to saturate SA residues on the B16F10 cell surface. The results show that after blocking, the uptake of lip\RB\P18-APBA-HA is significantly reduced. This suggests that lip\RB\P18-APBA-HA is primarily responsible for APBA-SA mediated cellular uptake. Since the pH value of the tumor microenvironment is usually between 6.5 and 6.9, the phenylboronic acid ester bond of the P18-APBA-HA is broken under the action of the tumor tissue weak acid microenvironment and the SA on the surface of tumor cells. Therefore, we speculate that at the tumor site, the surface HA of lip\rb\p18-APBA-HA may be shed, leaving more APBA exposed on the liposome surface, restoring its ability to specifically target SA residues at the tumor site.
Example 7lip EPA P18 APBA-HA in vitro ROS production experiments
The experimental method comprises the following steps: B16F10 cells (6X 10) 3 Individual/well) was inoculated on a ViewPlate-96 microplate and incubated overnight with PBS, EPA+P18, lip\EPA\P18-APBA and Li, respectivelyTreatment with p\EPA\P18-APBA-HA (EPA 2.5. Mu.g/mL, P18.25. Mu.g/mL). After 4h incubation, cells were washed and incubated with DCFH-DA (10. Mu.M) in serum-free medium at 37℃for 30min in the dark. A portion of the cells was irradiated with 671nm laser (0.8W/cm) 2 2 min). Subsequently, the cells were washed, fixed and stained. Finally, intracellular ROS production was observed using an operatta CLS high content imaging system.
Experimental results: as shown in FIG. 7, only weak fluorescent signals were detected by PBS control, EPA+P18, and Lip\EPA\P18-APBA-HA cells. After directing laser irradiation of the epa+p18 group, P18 in the ground state was stimulated to jump and interact with oxygen, and an increase in ROS signaling was observed in tumor cells. After illumination of the Lip\EPA\P18, lip\EPA\P18-APBA and Lip\EPA\P18-APBA-HA groups, significant ROS signals were also detected in the cells. In addition, after illumination, the ROS signals in the Lip\EPA\P18-APBA and Lip\EPA\P18-APBA-HA group cells were higher, possibly due to the enhancement of drug uptake by B16F10 cells.
Example 8 in vitro cytotoxicity Studies of lip/EPA/P18-APBA-HA
The experimental method comprises the following steps: B16F10 cells (5X 10) 3 Individual/well) were inoculated in 96-well plates and cultured overnight. Then treated with a range of drug concentrations of P18, EPA+P18, lip\EPA\P18-APBA and lip\EPA\P18-APBA-HA, respectively. After incubation for 4h, a portion of the cells was treated with laser light (671 nm, 0.8W/cm) 2 2 min). The medium was discarded and replaced with 100. Mu.L of serum-free medium (containing 10. Mu.L of CCK-8) and incubated for 2h. The optical density was measured by an enzyme-labeled instrument and the cell viability was calculated.
Experimental results: we studied the dark and phototoxicity of free P18 to tumor cells at different concentrations (fig. 8A). Under the condition of no light, the free P18 has no obvious inhibition effect on tumor cells in a certain concentration range, which indicates that the P18 has low toxicity and good biocompatibility. The differences were not significant after addition of EPA after light irradiation, indicating that EPA did not induce cytotoxicity. After illumination, the killing effect of epa+p18l+ on tumor cells increased significantly in concentration dependence (fig. 8B). Furthermore, lip\epa\p18-apbal+ and lip\epa\p18-APBA-HA l+ treatment induced more cell death than the free drug group, possibly due to enhanced cellular uptake. According to previous experiments, both free P18 and P18-loaded liposomes have photothermal properties and are capable of producing ROS in cells. Thus, when laser light irradiates cells of internalized P18 and P18 liposomes, a PDT/PTT combined effect is produced, enhancing the killing effect on tumor cells.
Example 9 in vitro induced ICD experiments with lip/EPA/P18-APBA-HA
The experimental method comprises the following steps: extracellular HMGB1 and ATP levels were detected with HMGB1 ELISA assay kit and chemiluminescent ATP detection kit, respectively. Briefly, B16F10 cells were attached and then treated with PBS, EPA+P18, lip\EPA\P18-APBA or lip\EPA\P18-APBA-HA (EPA 2.5. Mu.g/mL, P18.25. Mu.g/mL) for 4h, respectively, and then the cells were washed and added to fresh medium. Exposing a portion of the cells to a power density of 0.8W/cm 2 Is irradiated with a 671nm laser for 2min. After 24h incubation, cell supernatants were obtained for HMGB1 and ATP measurements according to the manufacturer's protocol.
Experimental results: the photoimmunotherapy induces ICD, thereby releasing DAMPs as dangerous signals to activate DCs cells, thereby stimulating tumor antigen presentation to T cells and eliciting tumor immune responses. DAMPs include HMGB1 released by tumor cells and exocrine ATP. Thus, we first assessed ICD of B16F10 tumor cells induced in vitro for each dosage form group by detecting extracellular HMGB1 and ATP levels. As shown in fig. 9, none of the non-laser irradiated formulation groups caused an increase in extracellular HMGB1 or ATP secretion. After laser irradiation, extracellular HMGB1 or ATP secretion was increased to a different extent for each formulation group. In particular, the lip\EPA\P18-APBA L+ and lip\EPA\P18-APBA-HA L+ treatment groups can more significantly promote up-regulation of extracellular HMGB1 and ATP levels.
Example 10 tissue distribution of lip/EPA/P18-APBA-HA
The experimental method comprises the following steps: to establish a B16F10 tumor-bearing mouse model, the right anterior axilla of female C57BL/6 mice was subcutaneously injected 5.0X10 5 B16F10 cells were maintained to tumor volume suitable for the experiment. The B16F10 tumor-bearing mice were randomly divided into 4 groups (n=3), and the tail vein injection was performed with cyanine 5.5 (cy 5.5) labeled liposomes lip\cy5.5\p18, lip\cy5.5\p18-APBA and lip\cy5.5\p18-APBA-HA and Free cy5.5 solutions (cy 5.5 2mg/kg, P18.6 mg °f-kg). Mice were sacrificed at 24h and heart, liver, spleen, lung, kidney and tumor tissues were taken, recorded and imaged.
Experimental results: as a result, as shown in FIG. 10, free Cy5.5 was mainly distributed in the liver and kidney, and a certain fluorescence signal was also present in the tumor site. However, the fluorescence signal intensity was lower for the Free cy5.5 group than for the cy5.5 labeled liposome group at each tissue, organ and tumor site. In addition, the fluorescence signal of the cy 5.5-labeled liposome group was not only distributed in the liver, kidney and spleen, but also the signal at the tumor site was more remarkable. Notably, the local fluorescence signal of lip\cy5.5\p18-APBA-HA in tumors was significantly stronger than that of lip\cy5.5\p18-APBA group and the distribution in liver, kidney, spleen was higher. The lip\Cy5.5\P18-APBA-HA HAs high targeting property to tumor tissues, probably because the lip\Cy5.5\P18-APBA-HA can play a long circulation role under the protection of an HA hydrophilic shell, so that nano particles leak into a tumor neovascular orifice (with the diameter of 200-600 nm) through blood circulation and accumulate in the tumor by utilizing the EPR effect. After the in vitro uptake result is combined and the HA targeting nano drug delivery system enters the extracellular space of tumor tissue, the surface HA can be removed, and the phenylboronic acid group which can be specifically identified by the SA on the surface of the melanoma cell is exposed, so that the specific uptake of the melanoma cell to the nano particle is further enhanced.
Example 11 in vivo antigen-induced tumor therapeutic Effect of lip/EPA/P18-APBA-HA
The experimental method comprises the following steps: b16F10 tumor-bearing mice were randomly divided into 7 groups (n=6), and tail vein injection of PBS, epa+p18, lip\epa\p18-APBA and lip\epa\p18-APBA-HA (EPA 3mg/kg, p180.6mg/kg). After 12h, the sample was irradiated with a 671nm laser (0.8W/cm) 2 2 min) irradiating the tumor site. Mice were treated once every 2 days, and mice body weight and tumor volume were measured every other day during treatment. On day 14, all mice were sacrificed and tumors and Tumor Draining Lymph Nodes (TDLNs) were collected and blood was further analyzed.
Experimental results: the results are shown in fig. 11, where the tumor volume of the PBS group increased rapidly, demonstrating successful model establishment. The free EPA+P18 group had weak inhibitory effect on tumor growth in the early stage (within 6 days), and the tumor growth was restored to rapid growth in the latter stage. The therapeutic effect of the EPA+P18L+ group was slightly improved compared to the free EPA+P18 group. The overall efficacy of the photo-immune treatment group was significantly better than the other non-illuminated groups. Wherein, the growth rate of the tumor volume of the Lip\EPA\P18-APBA L+ treatment group and the Lip\EPA\P18-APBA-HA L+ treatment group is slower within 14 days. On day 14, the tumor inhibition rates of the lip\EPA\P18-APBA L+ treatment group and the lip\EPA\P18-APBA-HA L+ treatment group were 91.32% and 95.45%, respectively, without significant differences.
EXAMPLE 12 in vivo induced DCs maturation assay of lip/EPA/P18-APBA-HA
The experimental method comprises the following steps: on day 14, mice were sacrificed, DCs in TDLNs were collected and stained, and mature dendritic cells (mDCs) were analyzed by flow cytometry. Expression of CD40, CD80 and CD86 markers in mDCs was used to quantify the maturity results of DCs. Briefly, excised TDLNs were ground and centrifuged to obtain a cell suspension. Staining with anti-CD 11c-FITC, anti-CD 40-PE, anti-CD 80-PE and anti-CD 86-APC antibodies followed by detection by flow cytometry.
Experimental results: as shown in FIG. 12, EPA+P18 and lip\EPA\P18-APBA-HA treatment groups slightly stimulated tumor cells to release TAAs, resulting in maturation of DCs. Interestingly, there was a significant increase in the maturation of DCs in all groups under light irradiation, especially in the lip\epa\p18-APBA-HA l+ treated group. After Lip\EPA\P18-APBA-HA reaches the tumor site, more target ligand can be exposed through pH response transformation, so that internalization of tumor cells is promoted, and DCs maturation is better promoted.
Example 13 in vivo IDO inhibition experiment of lip/EPA/P18-APBA-HA
The experimental method comprises the following steps: the tumor tissue homogenate of the mice is taken after the treatment. After centrifugation at 3000 Xg for 10min, 100. Mu.L of tumor tissue homogenate supernatant was taken. Expression of Trp and Kyn was then detected using the Elisa kit.
Experimental results: as a result, as shown in FIG. 13, free EPA+P18 or EPA+P18L+ was able to slightly down-regulate the Kyn/Trp ratio. Due to liposome-mediated tumor targeting, drug concentration at the tumor site is increased. Thus, the Kyn/Trp ratio was significantly reduced for each drug loaded liposome group. Interestingly, lip\EPA\P18-APBA-HAL+ HAs the best inhibitory effect on IDO, which is probably due to the layered targeting design of the dual drug delivery system, enabling lip\EPA\P18-APBA-HA to be better internalized into tumor cells and release the drug under laser irradiation. Thus, the inhibition of IDO in tumor cells by EPA of the lip\EPA\P18-APBA-HAL+ treatment group can be better exerted.
Example 14lip EPA P18 APBA-HA in vivo promotion of immune-related cytokine secretion
The experimental method comprises the following steps: immune-related cytokines also play an important role in anti-tumor immune responses, where Th 1-type cytokines mediate mainly cellular immune responses and promote killing of CTLs, such as interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma), among others. Thus, we collected serum from mice after various treatments and analyzed the serum for IL-6, TNF-. Alpha.and IFN-. Gamma.using ELISA kits according to the manufacturer's protocol.
Experimental results: the results are shown in fig. 14, where the drug loaded liposome group showed higher immunity intensity than the free drug group. Furthermore, the highest level of cytokine secretion in the lip\EPA\P18-APBA-HA L+ treated group indicated successful induction of the immune response.
Example 15 experiments on in vivo activation of Ths, CTLs and inhibition of Tregs by lip/EPA/P18-APBA-HA
The experimental method comprises the following steps: CTLs can directly kill tumor cells, helper T cells (Ths) are critical in the immunomodulation process, and CTLs and Ths play an important role in anti-tumor immune responses. In contrast, tregs negatively regulate the immune response of the body and help tumor cells evade immune surveillance. Thus, we collected tumors were also used to analyze the infiltration of intratumoral T lymphocytes and analyzed by flow cytometry. Briefly, tumor tissue is ground, lysed and centrifuged to obtain a cell suspension. Analysis of the lymphocytes from tumors by staining them with anti-CD 3-PAC and anti-CD 4-FITC according to the protocol (CD 3 + CD4 + ). Analysis of CTLs (CD 3) by anti-CD 3-APC and anti-CD 8a-PE antibodies + CD8 + ) Tregs (CD 4) analysis by anti-CD 4-FITC and anti-Foxp 3-PE antibodies + Foxp3 + )。
Experimental results: as shown in fig. 15, CTLs and Ths increased and Tregs decreased in each treatment group compared to PBS group. Among them, the lip\EPA\P18-APBA-HAL+ treatment group had the highest CTLs and Ths levels and the lowest Tregs levels. The results show that lip\EPA\P18-APBA-HA can effectively ablate primary tumors and excite strong anti-tumor immunity under the irradiation of laser.
Example 16lip EPA P18 APBA-HA in vivo anti-tumor pulmonary metastasis efficacy
The experimental method comprises the following steps: to study the inhibition of Lip\EPA\P18-APBA-HA on melanoma metastasis, the tumor volume of mice with B16F10 tumor-bearing model reached 90mm 3 At this time, each mouse was injected with B16F10 cells (5X 10) 5 And/or just). The mice were then randomized into 7 groups (n=6) and given the same treatment as B16F10 tumor-bearing mice. On day 14, all mice were sacrificed, lungs were excised, imaged and subjected to histopathological staining analysis.
Experimental results: as a result, as shown in fig. 16, on day 14, all lung tissue structures of the PBS group were destroyed and the alveoli disappeared. In EPA+P18 group and lip\EPA\P18-APBA-HA group, tumor tissue grows in an infiltrative manner, and part of lung tissue structure is destroyed. Smaller transfer surfaces were observed after illumination, with the transfer-resistant effect of the lip\EPA\P18-APBA-HAL+ group being most pronounced. It is believed that this layered targeted dual drug delivery system greatly requires the combination of EPA and P18 to achieve effective treatment of melanoma metastasis.
Claims (10)
1. A layered targeting dual delivery system based on photo-immunotherapy, which is characterized in that the layered targeting dual delivery system is mainly formed by preparing a complex from a photosensitizer, a phenylboronic acid derivative and hyaluronic acid, then inserting the complex into a phospholipid membrane formed by phospholipid and cholesterol, and finally encapsulating an IDO inhibitor.
2. The layered targeted dual delivery system based on photo-immunotherapy of claim 1, wherein the photosensitizer is selected from the group consisting of rhodopsin 18; the IDO inhibitor is selected from one of indoximod, NLG919 and Epacadostat (EPA).
3. The layered targeted dual delivery system based on photo-immunotherapy according to claim 1, wherein the phenylboronic acid derivative is selected from 4-aminomethylphenylborates; the molecular weight of the hyaluronic acid is 5000Da-10000Da; the phospholipid is one of natural phospholipid, soybean lecithin and egg yolk lecithin.
4. A method of preparing a layered targeted dual delivery system based on photo-immunotherapy as claimed in any one of claims 1-3, comprising the steps of:
(1) Dissolving a photosensitizer and a condensing agent, activating, adding phenylboronic acid derivatives in a stirring state, and stirring at room temperature for reaction to obtain a photosensitizer-phenylboronic acid compound;
(2) Respectively dissolving hyaluronic acid and photosensitizer-phenylboronic acid compound, dropwise adding photosensitizer-phenylboronic acid compound solution into hyaluronic acid solution under stirring, adjusting pH, and stirring at a certain temperature for reaction to obtain photosensitizer-phenylboronic acid-hyaluronic acid compound;
(3) Taking phospholipid and cholesterol, preparing a lipid film, adding a solution containing a photosensitizer-phenylboronic acid-hyaluronic acid compound for hydration, and performing ultrasonic treatment to obtain a compound-liposome solution;
(4) And (3) dropwise adding the solution containing the IDO inhibitor into the complex-liposome solution under the stirring condition, incubating, and removing unencapsulated IDO inhibitor to obtain the layered targeted dual delivery system.
5. The method of preparing a layered targeted dual delivery system based on photo-immunotherapy according to claim 4, wherein in step (1), the condensing agent is selected from one or more of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4-Dimethylaminopyridine (DMAP) and 1-Hydroxybenzotriazole (HOBT); the mol ratio of the photosensitizer to the phenylboronic acid derivative is 1 (2-4); the reaction time is 22-26h.
6. The method of claim 4, wherein in step (2), the molar ratio of photosensitizer-phenylboronic acid complex to hyaluronic acid is (4-6): 1; the pH is adjusted to 8-10; the reaction temperature is 60-70 ℃, and the reaction time is 22-26h.
7. The method of claim 4, wherein in step (3), the lipid film is prepared by a film dispersion method; the mass ratio of the phospholipid to the cholesterol is (8-10): 1; the mass ratio of the phospholipid to the photosensitizer-phenylboronic acid-hyaluronic acid complex is (3-5) 1; the solution containing photosensitizer-phenylboronic acid-hyaluronic acid complex is prepared by adopting (NH) 4 ) 2 SO 4 Solution of (NH) 4 ) 2 SO 4 The concentration of the solution is 200-300mM; the hydration temperature is 30-50 ℃, and the hydration time is 50-70min; the ultrasonic treatment adopts probe ultrasonic treatment, the ultrasonic power is 250-350W, and the ultrasonic treatment time is 4-6min; removing (NH) by dialysis after ultrasonic treatment 4 ) 2 SO 4 。
8. The method for preparing a layered targeted dual delivery system based on photo-immunotherapy according to claim 4, wherein in step (4), the mass ratio of IDO inhibitor to photosensitizer is (4-6) 1; the incubation temperature is 30-50 ℃, and the incubation time is 20-40min.
9. Use of a layered targeted dual delivery system based on photo-immunotherapy as claimed in any one of claims 1-3 for the preparation of a medicament for the treatment of melanoma.
10. The use according to claim 9, wherein the medicament for treating melanoma is capable of inhibiting proliferation and metastasis of melanoma.
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