Double-response cascade cross-linked hydrogel for targeting lung cancer and preparation method of double-response cascade cross-linked hydrogel

Abstract

The invention relates to the technical field of biomedical materials, and provides dual-response cascade cross-linked hydrogel for targeting lung cancer. The hydrogel comprises a phase A and a phase B, wherein the phase A comprises hyaluronic acid-phenylboronic acid and four-arm polyethylene glycol-sulfydryl; and the phase B comprises polyethylene glycol diacrylate, a photoinitiator LAP and functionalized up-conversion nanoparticles loaded with Tasqueinimod, a PD-L1 antibody and an MMP-2 sensitive peptide. The hydrogel forms gel preliminarily through acid response to prevent diffusion, cross-linking shaping is enhanced by means of light triggering, unification of injectability and high mechanical strength is achieved, meanwhile, through a sequential control release strategy, a PD-L1 antibody is rapidly released to activate immune response, Tasqueinimod is slowly released to block immunosuppression, and drug antagonism is avoided. The hydrogel is high in intervention precision and high in positioning performance in a precise injection mode that a bronchoscope ultrasonically guides a bronchus wall penetrating needle to suck a biopsy channel, and efficient targeted therapy of the lung cancer is expected to be achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological nanotechnology, and particularly relates to a dual-response cascade cross-linked hydrogel for targeting lung cancer and a preparation method thereof. BACKGROUND

[0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality worldwide, which seriously threatens human health. Due to its atypical early symptoms, most patients are in the middle and late stages when diagnosed, missing the best opportunity for surgical treatment, and clinical treatment relies on non-surgical means such as chemotherapy, targeted therapy and immunotherapy. However, lung cancer has strong tissue heterogeneity and complex tumor microenvironment, and traditional drug delivery methods have the problems of large systemic toxicity, insufficient drug concentration at the tumor site, short action time and the like, resulting in unsatisfactory treatment effect. Especially in the process of multi-drug combination therapy, the release rate and action target of different drugs in the body are inconsistent, which easily produces antagonism or enhances toxicity, seriously affecting the safety and effectiveness of treatment. Therefore, developing a new treatment strategy that can realize the directional delivery and time-controlled release of drugs is of great significance for improving the treatment efficiency of lung cancer, reducing side effects and prolonging the survival period of patients.

[0003] At present, in order to solve the problems of drug targeting delivery and time-controlled release in lung cancer treatment, the existing technology has developed various schemes such as nano-drug delivery system, intelligent response material and local drug delivery technology. Among them, injectable hydrogel has attracted much attention due to its good local retention capacity. However, the pH-responsive injectable hydrogel and self-healing injectable hydrogel in current research still have two major technical bottlenecks: on the one hand, the injectability and in-situ mechanical stability of the material are difficult to balance, resulting in easy damage or displacement of the structure after implantation; on the other hand, the drug release relies on a single response mechanism, which cannot realize the time-controlled release of “rapid activation and sustained inhibition” required by multi-drug synergy, seriously limiting the effectiveness of the combined treatment strategy. Therefore, the existing technology still cannot fully meet the comprehensive needs of precision, time-controlled, multi-drug synergy and local stable release in lung cancer treatment, and new breakthroughs are urgently needed. SUMMARY

[0004] The purpose of the present application is to provide a dual-response cascade cross-linked hydrogel for targeting lung cancer to solve the above technical problems.

[0005] The hydrogel of the present application comprises phase A and phase B, phase A comprises hyaluronic acid-benzene boronic acid (HA-SBA) and four-arm polyethylene glycol-thiol; phase B comprises polyethylene glycol diacrylate, a photoinitiator LAP and PTZ@UCNP.

[0006] Specifically, PTZ@UCNP comprises Tasquinimod, ZIF-8, Nd 3+Sensitized NaYF4:Yb, Tm upconversion nanoparticles (UCNPs), PD-L1 antibody and MMP-2 sensitive peptide (GPLGLG).

[0007] Specifically, the mass ratio of hyaluronic acid-boronic acid, four-arm polyethylene glycol-mercapto, polyethylene glycol diacrylate, photoinitiator LAP and PTZ@UCNP in the hydrogel of the application is 20:20:100:1:4.

[0008] The application also provides a double-response cascade cross-linked hydrogel for targeting lung cancer and a preparation method thereof, specifically comprising the following steps:

[0009] (4) Preparation of PTZ@UCNP: 0.1M zinc nitrate solution and 0.8M 2-methylimidazole solution were mixed, Tasquinimod was added, and ZIF-8 nanoparticles (Tas@ZIF-8) with a Tas loading rate of 15% (w / w) were synthesized by one-pot method, and then washed and dried by centrifugation at 12000 rpm for 10 min. Subsequently, the Tas@ZIF-8 was mixed with 2mg / mL Nd 3+ Sensitized NaYF4:Yb, Tm upconversion nanoparticles were dispersed in a PBS / ethanol mixed solution with a volume ratio of 9:1, and were coated with 1mg / mL PD-L1 antibody by using a nanoprecipitation method to embed the UCNPs in the PLGA layer. Finally, the MMP-2 sensitive peptide was grafted onto the surface of the PLGA by using 0.2M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.05M N-hydroxysuccinimide (NHS), and PTZ@UCNP was obtained.

[0010] (5) Preparation of mixed precursors: 10mg / mL HA-SBA and 10mg / mL four-arm polyethylene glycol-mercapto were dissolved in sterile PBS with pH 7.4, uniformly stirred and sterilized by 0.22μm filtration to obtain phase A, which was stored at 4℃ for standby. 5% (w / v) polyethylene glycol diacrylate (PEGDA), 0.5mg / mL photoinitiator LAP and PTZ@UCNP were dispersed in sterile PBS with pH 7.4 to obtain phase B, which was stored at 4℃ under light-proof conditions for standby.

[0011] (6) Preparation of double-barrel syringe injection device: a double-barrel syringe was used to take phase A and phase B respectively, and the output was connected to a puncture needle for transbronchial needle aspiration (TBNA) guided by bronchoscope ultrasound, so as to realize precise injection at the tumor site.

[0012] Further, the final concentration of Tasquinimod in step (1) is 10mg / mL.

[0013] Further, the volume ratio of the PBS / ethanol mixed solution in step (1) is 9:1.

[0014] Further, the MMP-2 sensitive peptide in step (1) is GPLGLG.

[0015] Further, the concentration of the PTZ@UCNP in step (2) is 2 mg / mL.

[0016] Further, the puncture needle in step (3) is 25G.

[0017] The application also provides a use of the dual-response cascade cross-linked hydrogel for targeting lung cancer in the preparation of a drug for lung cancer treatment, characterized in that the drug is configured to be cured by light cross-linking after being administered to a lesion, and needs to be irradiated for 10 seconds by an 808 nm near-infrared laser with a power density of 1 W / cm 2 .

[0018] The application has the following advantages:

[0019] 1. The application prevents diffusion by acid-responsive preliminary gelation and resists stress by light-triggered reinforcement, synchronously realizing the injectability and high mechanical strength of the material, effectively overcoming the contradiction in performance of traditional gels, and forming a "positioning-release-reinforcement" integrated treatment strategy.

[0020] 2. The application adopts a time sequence controlled release strategy, realizes rapid release of PD-L1 antibody to activate immune response, and effectively blocks immune suppression with the help of Tasquinimod sustained release, avoids drug antagonism through time sequence separation, and significantly improves the anti-tumor effect.

[0021] 3. The application realizes precise positioning of the hydrogel by bronchoscopy under the guidance of ultrasound through the transbronchial wall needle aspiration channel, and has the advantages of high intervention precision and high positioning. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the rheological property characterization of the hydrogel of the application.

[0023] Figure 2 is the PD-L1 drug release performance characterization of the hydrogel of the application.

[0024] Figure 3 is the Tasquinimod drug release performance characterization of the hydrogel of the application.

[0025] Figure 4 is the in vivo retention rate characterization of the hydrogel of the application.

[0026] Figure 5 is the anti-tumor performance characterization of the hydrogel of the application.

[0027] Figure 6 is a biological safety performance table of the hydrogel of the present application. DETAILED DESCRIPTION

[0028] The technical solutions described in the present application will be clearly and completely described below in combination with the accompanying drawings in the embodiments of the present application. Obviously, the embodiments described in the present application are only a part of the feasible technical solutions of the present application, and other embodiments obtained by those skilled in the art on the basis of the embodiments of the present application without any creative labor should be regarded as belonging to the protection scope of the present application.

[0029] Example 1: Preparation of dual-responsive cascade cross-linked hydrogel

[0030] (1) Preparation of PTZ@UCNP: 0.1M zinc nitrate solution was mixed with 0.8M 2-methylimidazole solution, and Tasquinimod was added to a final concentration of 10mg / mL, and Tas@ZIF-8 with a Tas loading rate of 15%(w / w) was synthesized by one-pot method, and was washed and dried by centrifugation at 12000rpm for 10min. Subsequently, Tas@ZIF-8 was mixed with 2mg / mL Nd 3+ The UCNPs of sensitized NaYF4:Yb,Tm were dispersed in a mixed solution of PBS / ethanol with a volume ratio of 9:1, and were coated with PLGA containing 1mg / mL PD-L1 antibody by nano-precipitation method, so that the UCNPs were embedded in the PLGA layer. Finally, the MMP-2 sensitive peptide (GPLGLG) was grafted on the surface of PLGA by 0.2M EDC and 0.05M NHS, and PTZ@UCNP was obtained.

[0031] (2) Preparation of mixed precursors: 10mg / mL HA-SBA and 10mg / mL four-arm polyethylene glycol-thiol were dissolved in sterile PBS with pH 7.4, and were fully stirred and sterilized by 0.22μm filtration, to obtain phase A, which was stored at 4℃ for standby. 5%(w / v) PEGDA, 0.5mg / mL photoinitiator LAP and 2mg / mL PTZ@UCNP were dispersed in sterile PBS with pH 7.4 to obtain phase B, which was stored at 4℃ under light-proof conditions for standby.

[0032] (3) Preparation of dual-cylinder injection device: a dual-cylinder syringe was used to take phase A and phase B respectively, and was output through a static mixing needle cylinder, and the outlet was connected to a 25G puncture needle of TBNA, so as to realize precise injection at the tumor site under the guidance of bronchoscopy ultrasound.

[0033] Comparative Example 1: Preparation of acid-unresponsive hydrogel

[0034] The difference between this comparative example and Example 1 is that there is no HA-SBA in phase A.

[0035] Preparation of non-optical responsive hydrogel

[0036] This comparative example differs from Example 1 in that no UCNP is added in the system.

[0037] Preparation of MMP-2 enzyme responsive cascade crosslinked hydrogel

[0038] This comparative example differs from Example 1 in that no MMP-2 sensitive peptide (GPLGLG) is contained.

[0039] Preparation of mixed cascade crosslinked hydrogel

[0040] This comparative example differs from Example 1 in that phase A and phase B are pre-mixed before injection, instead of mixing at the moment of injection by a double-barreled device.

[0041] Preparation of non-responsive drug release cascade crosslinked hydrogel

[0042] This comparative example differs from Example 1 in that Lyso-AuNCs in the hydrogel layer are replaced by Lyso.

[0043] Preparation of Tasquinimod-unloaded cascade crosslinked hydrogel

[0044] This comparative example differs from Example 1 in that no Tasquinimod is loaded in the system.

[0045] Preparation of PD-L1 antibody-unloaded cascade crosslinked hydrogel

[0046] This comparative example differs from Example 1 in that no PD-L1 antibody is loaded in the system.

[0047] Test Example 1: Rheological characterization of the dual-responsive cascade crosslinked hydrogel prepared in Example 1

[0048] The gelation process of the hydrogels prepared in Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 4 was characterized using a rotational rheometer. Equal volumes of phase A and phase B were thoroughly mixed using a static mixer and then placed on the parallel plate fixture of the rheometer. The change in modulus with time was monitored under constant oscillatory strain (1%) and frequency (1 rad / s). The test was divided into two consecutive stages to mimic the in vivo environment: in the first stage, the mixed sample was exposed to PBS buffer at pH 6.5 for 10 min; in the second stage, the sample was irradiated with 808 nm near-infrared laser (power density 1 W / cm 2 ) for 10 s at the center region. The storage modulus (G’) of the sample was continuously monitored throughout the process.

[0049] Results are shown in Figure 1 Figure 6. The G' of the hydrogel prepared in Example 1 was 41.3 Pa and 904 Pa at stage one and stage two, respectively. The G' of the hydrogel prepared in Comparative Example 1 was 0.4 Pa at both stages. The G' of the hydrogel prepared in Comparative Example 2 was 42.4 Pa at both stages. The G' of the hydrogel prepared in Comparative Example 4 was 92.4 Pa and 913 Pa at stage one and stage two, respectively. This is because the hydrogel prepared in Example 1 has a weak but effective physical crosslinking network formed by the hydrophobic aggregation of HA-SBA after contacting the pH 6.3 buffer, which is sufficient to prevent premature diffusion before and after injection. Subsequently, a strong covalent crosslinking network is successfully constructed by the UCNPs-mediated photo-triggered thiol-ene "click" chemistry under 808 nm laser irradiation. The G' of Comparative Example 1 is extremely low due to the difficulty of hydrophobic aggregation of HA-SBA, and the serious loss of reactant diffusion seriously affects the formation of the second stage hydrogel. The G' of the hydrogel prepared in Comparative Example 2 is stagnant at a low level platform of the primary network, which indicates that the final high mechanical strength of the hydrogel depends on the secondary photo-crosslinking. The initial G' of Comparative Example 4 is significantly higher than that of other groups, which indicates that slow crosslinking occurs in the initial stage. In summary, the hydrogel prepared in Example 1 has good dual-responsive cascade crosslinking ability.

[0050] Test Example 2: Characterization of the drug release behavior of the dual-responsive cascade crosslinking hydrogel prepared in Example 1

[0051] To evaluate the drug release behavior of the hydrogels prepared in Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 5, the hydrogels were placed in 50 mL of pH 6.5 PBS medium (containing 100 ng / mL MMP-2 enzyme) at 37°C for drug release experiments. At 4 h, the experimental groups were irradiated with 808 nm near-infrared laser (1 W / cm 2 ) for 10 seconds. At 0.5, 1, 2, 4, 8 and 12 h, 3 mL of the test solution was sampled and an equal volume of fresh medium was supplemented at the same time. The collected samples were quantified for PD-L1 antibody concentration by enzyme-linked immunosorbent assay (ELISA), and Tasquinimod concentration was determined by high performance liquid chromatography (HPLC), and the cumulative release rate was calculated accordingly.

[0052] Results are shown in Figure 2 The cumulative release rates of PD-L1 in Example 1, Comparative Example 2 and Comparative Example 5 were 77%, 74% and 89% at 4 h, respectively, and all reached more than 90% at 12 h. The release rate of Comparative Example 3 was significantly lower, only 14% at 4 h and 21% at 12 h. Figure 3As shown, the cumulative release rate of Tasquinimod at 4h was 12% for Example 1, 42% for Comparative Example 2, and as high as 91% for Comparative Example 5; at 12h, Example 1 was only 36%, while Comparative Example 2 and Comparative Example 5 reached 88% and 94%, respectively. The PD-L1 release rates of Example 1, Comparative Example 2, and Comparative Example 5 were all over 74% within 4h, while Comparative Example 3 released very low, indicating that MMP-2 enzyme cutting was the key to triggering the rapid release of PD-L1. In addition, after near-infrared light irradiation, the release of Tasquinimod in Example 1 was significantly delayed, with a cumulative release rate of only about 36% at 12h, much lower than that of Comparative Example 2 (88%) and Comparative Example 5 (94%). The above results show that the hydrogel of Example 1 can achieve rapid release of PD-L1 and slow release of Tasquinimod, showing good time-controlled release ability.

[0053] Test Example 3: In vivo retention performance characterization of the dual-responsive cascade cross-linked hydrogel prepared in Example 1

[0054] To quantitatively evaluate the in vivo retention performance of the hydrogels prepared in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 4, we first chemically coupled the near-infrared fluorescent dye Cy5.5 to the PLGA shell of the PTZ@UCNP nanoparticles, so as to accurately track the nano-carriers by fluorescence signal. After the tumor-bearing mice were randomly divided, an equal dose of Cy5.5-labeled hydrogel was injected into the lung cancer site through the TBNA channel, and 808 nm near-infrared laser (1 W / cm2) irradiation was applied to the experimental groups for 10 seconds at 4h. At 1h, 24h, 72h, and 168h (7 days) after injection, whole-body images were collected using a small animal live imaging system, and the average fluorescence intensity of the tumor region was quantitatively analyzed. Taking the fluorescence intensity of the tumor at 1h after injection as the 100% benchmark, the tumor retention rate (%) at each time point was calculated as (fluorescence intensity at current time point / fluorescence intensity at 1h) x 100%.

[0055] The results are shown in Figure 4 As shown, the tumor retention rate of Example 1 at 7 days was 95.7%, the tumor retention rates of Comparative Example 1 at 24h and 7 days were 46.3% and 2.2%, respectively, the tumor retention rates of Comparative Example 2 at 24h and 7 days were 95.1% and 71.8%, respectively, and the tumor retention rates of Comparative Example 4 at 24h and 7 days were 73.5% and 54.6%, respectively. The rapid diffusion of Comparative Example 1 was because there was no HA-SBA for hydrophobic aggregation, Comparative Example 2 was because the physical network relying only on HA-SBA primary cross-linking was difficult to meet the mechanical requirements of long-term retention, and the low retention rate of Comparative Example 4 was because the pre-mixing leading to early cross-linking completely destroyed the distribution pattern of the drug, thereby affecting its retention performance. In summary, the hydrogel of Example 1 has excellent initial anti-diffusion and long-term retention ability.

[0056] Test Example 4: In vivo anti-tumor performance characterization of the dual- response cascade crosslinked hydrogel prepared in Example 1

[0057] To evaluate the anti-tumor effect of the hydrogels prepared in Example 1 and Comparative Examples 1-7, we established LLC lung cancer xenograft models in C57BL / 6 mice. When the tumor volume reached about 100 mm 3 Afterwards, the mice were randomly divided into 9 groups (n = 8) and injected with the corresponding preparation or PBS through the TBNA channel. 4 h after injection, the experimental groups were irradiated with 808 nm near-infrared laser (1 W / cm 2 ) for 10 seconds, and the tumor volume and body weight were regularly monitored within 50 days. At the end of the study, the mice were sacrificed, the tumors were weighed and analyzed.

[0058] The results are shown in Figure 5 Example 1 group had the lowest tumor weight (0.4 g), and the anti-tumor effect was significant, indicating that the timing strategy of rapid release of PD-L1 antibody and slow release of Tasquinimod effectively inhibited tumor growth. The tumor weight of Comparative Example 5 was 0.7 g, indicating that the timing release was better than the co-time release. The tumor weights of Comparative Examples 6 and 7 were 0.9 g and 1.2 g, respectively, and the therapeutic effect was lower than that of the combined preparation, highlighting the importance of drug synergy. The tumors in Comparative Examples 1-4 were all greater than 1.5 g, among which Comparative Example 1 had the worst effect, close to the blank group, confirming the necessity of the primary network for diffusion prevention; Comparative Examples 2 and 3 had weak anti-tumor effects, respectively demonstrating the key role of light-triggered anchoring and enzyme-controlled release in long-term slow release and rapid start of immunity; Comparative Example 4 had poor injectability and gel performance, which also led to a significant decrease in therapeutic effect. In summary, only Example 1 with a complete cascade response mechanism can achieve the optimal anti-tumor effect.

[0059] Test Example 5: Biocompatibility characterization of the dual-response cascade crosslinked hydrogel prepared in Example 1

[0060] To evaluate the biological safety of the hydrogel prepared in Example 1, the mice were euthanized on the last day of the experiment, and important organs (spleen, liver, heart, kidney, lung) were harvested. After H&E staining, the important organs were fixed, embedded and histologically examined. As shown in Figure 6 , we did not detect necrosis or obvious tissue damage (including apoptosis, nuclear fragmentation, pyknosis or inflammatory cell infiltration) in any treatment group. This indicates that the dose used is safe and does not have any obvious toxicity.

[0061] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and supplements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. A dual-responsive cascade cross-linked hydrogel targeting lung cancer, characterized in that, The hydrogel comprises phase A and phase B, phase A comprises hyaluronic acid-boronic acid, four-arm polyethylene glycol-thiol; phase B comprises polyethylene glycol diacrylate, photoinitiator LAP and PTZ@UCNP; The PTZ@UCNP comprises Tasquinimod, ZIF-8, Nd 3+ Sensitizing NaYF4:Yb, Tm upconversion nanoparticles, PD-L1 antibody and MMP-2 sensitive peptide The mass ratio of hyaluronic acid-boronic acid, four-arm polyethylene glycol-thiol, polyethylene glycol diacrylate, photoinitiator LAP and PTZ@UCNP in the hydrogel is 20:20:100:1:

4.

2. A method of preparing a dual-responsive crosslinked hydrogel targeting lung cancer according to claim 1, wherein, The preparation method comprises the following steps: (1) Preparation of PTZ@UCNP: 0.1M zinc nitrate solution and 0.8M 2-methylimidazole solution are mixed, Tasquinimod is added, and Tas@ZIF-8 with a Tas loading rate of 15% by mass fraction is synthesized by one-pot method, and is collected after centrifugation at 12000rpm for 10min, washing and drying; Subsequently, Tas@ZIF-8 was mixed with 2 mg / mL Nd 3+ The sensitized NaYF4:Yb, Tm upconversion nanoparticles were dispersed in a mixed solution of PBS / ethanol with a volume ratio of 9:1, and were coated with PLGA containing 1 mg / mL PD-L1 antibody by the nanoprecipitation method to embed the upconversion nanoparticles in the PLGA layer; finally, the MMP-2 sensitive peptide was grafted onto the surface of the PLGA by 0.2M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 0.05M N-hydroxysuccinimide to obtain PTZ@UCNP; (2) Preparation of mixed precursors: 10mg / mL HA-SBA and 10mg / mL four-arm polyethylene glycol-thiol are dissolved in sterile PBS with pH 7.4, uniformly stirred and sterilized by 0.22μm filtration to obtain phase A, which is stored at 4℃ for standby; 5% by mass polyethylene glycol diacrylate, 0.5mg / mL photoinitiator LAP and PTZ@UCNP are dispersed in sterile PBS with pH 7.4 to obtain phase B, which is stored at 4℃ under light-proof condition for standby; (3) Preparation of double-barrel syringe device: phase A and phase B are taken by double-barrel syringes respectively, and are output through a static mixing needle barrel, and the outlet is connected to a puncture needle for bronchoscopic ultrasound-guided transbronchial needle aspiration to realize precise injection at the tumor site.

3. The method of claim 2, wherein the hydrogel is prepared by the method comprising the steps of: The final concentration of Tasquinimod in step (1) is 10mg / mL.

4. The method of claim 2, wherein the hydrogel is prepared by the method comprising the steps of: The volume ratio of the PBS / ethanol mixed solution in step (1) is 9:

1.

5. The method of claim 2, wherein the hydrogel is prepared by the method comprising the steps of: The MMP-2 sensitive peptide in step (1) is GPLGLG.

6. The method of claim 2, wherein the hydrogel is prepared by the method comprising the steps of: The concentration of PTZ@UCNP in step (2) is 2mg / mL.

7. The method of claim 2, wherein the hydrogel is prepared by the method comprising the steps of: The puncture needle in step (2) is 25G.

8. Use of the hydrogel according to claim 1 in the manufacture of a medicament for the treatment of lung cancer. The drug is configured to be photo-crosslinking cured upon administration to the lesion by irradiation with 808 nm near-infrared laser at a power density of 1 W / cm 2 for 10 seconds.

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