Structural Modification Of Saha And Its Anti-tumor Effect Of Folate Targeting Nanohychogel

Histone deacetylase inhibitors（HDACIs）are a class of anticancer drugs used to treat non-solid tumors such as lymphoma,leukemia,blood diseases,etc.One of the representative octadecanoic acid（SAHA）was marketed in the United States in 2016.This anticancer drug has a good effect in the treatment of non-solid tumors.However,due to many clinical shortcomings of SAHA,this article intends to use structural optimization methods to improve it.Therefore,three highly soluble SAHA precursor compounds were designed and synthesized.After optimizing the structure of SAHA,there are still some shortcomings,such as the unsatisfactory effect of tumor cell apoptosis and drug resistance.Studies have shown that HDACIs is resistant to the treatment of tumors.It is difficult for a single HDACIs to achieve a therapeutic effect on tumors.Therefore,we want to use a combination of chemotherapy and HDACIs to achieve better results for tumor treatment.As a new type of minimally invasive anti-tumor therapy,photodynamic therapy（PDT）,despite its rapid development,still has some shortcomings.After PDT treatment,it changes the tumor microenvironment and affects the survival of tumor cells,such as cells.Differentiation,proliferation,angiogenesis,and apoptosis,and abnormal levels of histone acetylation in cells after photodynamic therapy.Studies have shown that HDACIs can reduce the activity of histone deacetylase in tumors after photodynamic therapy.The combination of HDACIs and PDT is a necessary means to improve the efficacy of PDT.In order to overcome the shortcomings of the anti-tumor drug SAHA,such as poor solubility,short half-decline and low cell intake,we designed and synthesized a nano-hydrogel drug delivery system with folic acid targeting and SAHA via ester bond through a nano-carrier to improve these shortcomings.The nano-hydrogel system has good water solubility,can evade immune system clearance,and has the characteristics of long circulation.Under the slightly acidic environment of tumor site,the ester bond breaks,the anti-tumor drug SAHA is released,and the anti-tumor effect is realized.The drug loading system is evaluated through in vivo and in vitro experiments.1 Synthesis,characterization and activity of HDACIs class small molecule compoundsFirstly,we try to improve the poor solubility of SAHA by using small molecular active groups.We connect ethyl 4-aminobutyrate hydrochloride,ethyl 5-aminovalerate hydrochloride and ethyl 6-aminocaproate hydrochloride to the hydroxyl group of SAHA by using the condensation agent HATU.The results show that the synthesis of the three compounds is successful.We also use melanoma cell line to study the effect the toxicity of three combinations was tested.In the synthesis of bimodal antitumor drugs,the photosensitizer dihydroporphine e 6 was used as the raw material,and dihydroporphine e4 was obtained by reflux at 110℃in pyridine.The17 carboxyl group of dihydroporphine e4 was linked to the small molecule active group by amidation reaction.A new HDACIs small molecule compound with hydroxamic acid structure was obtained by connecting cinnamic acid,the active group of aromatic small molecule compound,and then reacting with fresh saturated hydroxylamine hydrochloride.Another compound was synthesized by one-step alkaline hydrolysis to form carboxyl group after the small molecule active group was attached at the17 position of e4,and then o-phenylenediamine was connected through the condensation of amide bond to synthesize a HDACIs small molecule compound similar to sidaaniline.According to the analysis of the results of NMR and MS,we have successfully synthesized two new HDACIs small molecular compounds with dual-mode antitumor activity.In order to solve the problem of poor solubility of drugs,it is proposed to synthesize a new carrier or group modification to improve the disadvantages of drugs,so the second part of this paper is carried out.2 Synthesis,characterization and activity of folate-targeted bovine serum albumin nanohydrogels.An attempt was made to connect the SAHA and the albumin-encapsulated nanohydrogel through an ester bond,while using folic acid as a target on the surface of the albumin nanohydrogel to improve the targeting of the drug delivery system.The poorly soluble drug SAHA is connected to the surface of the nanohydrogel to form a layer of hydration film.Firstly,use NAS to modify the double bond on the surface of bovine serum albumin,add various monomer compounds,add cross-linking agent and polymer chain activator to obtain bovine serum albumin nanohydrogel,and use the condensing agent EDC/NHS to connect folic acid.On the surface of the nanohydrogel,using N,N-carbonyldiimidazole（CDI）as a condensing agent,forms a nanohydrogel drug-loading system（FVBN）with FA targeting.FVBN nanocarriers were characterized by IR and ¹H-NMR.The particle size,zeta potential,and drug loading of the nanohydrogel and the blank material were measured.The maximum drug loading of the nanohydrogel was 20.28%.The transmission electron microscope was used to observe the microstructures of the drug-loaded nanohydrogel and the blank material,showing an irregular spherical shape.The in vitro stability of nanohydrogels in PBS and serum was investigated.The in vitro stability studies found that drug-loaded nanohydrogels have good stability,and the particle size and PDI change within 48 hours are small.Secondly,the IC₅₀values of n BSA,SAHA,and FVBN to HUVEC,B16-F10,and A2780 were measured using in vitro cytotoxicity experiments.FITC was used to modify n BSA,VBN,and FVBN,and the modified n BSA,VBN,and FVBN showed green fluorescence under laser excitation.Nuclear nucleus was stained with DIPA,and the intensity of fluorescence intensity entering the cells was observed with a laser confocal microscope.Flow cytometry was used to quantitatively analyze the fluorescence intensity of n BSA,VBN,and FVBN,respectively.The effects of SAHA,VBN,and FVBN on cell apoptosis and cell cycle arrest were investigated by flow cytometry.Free SAHA,VBN,and FVBN block tumor cells at G1/G0.The maximum cycle block of free SAHA is 86.33%,and the maximum period blocks of tumor cells by VBN and FVBN are 89.16%and89.24%,respectively.Compared with SAHA,FVBN has obvious advantages in blocking tumor cells.In the end,this paper studied the anti-tumor effect of FVBN at the animal level.The tumor-bearing models of C57BL/6 mice and nude mice were evaluated for anti-tumor,and tumor models of mice were established.Set up normal saline group,SAHA group,VBN group,FVBN group,5mice in each group.SAHA 5 mg/kg was administered by tail vein injection once every 2 days for a total of 5 times.The tumor size and weight of the mice were measured every two days,and the survival of the mice was recorded.The lung tissue of each group of C57BL/6 mice was photographed and observed by H&E staining.Liver tissues of each group of nude mice were photographed,and heart,liver,spleen,lung,and kidney sections of nude mice were observed by H&E staining,showing that FVBN has a significant inhibitory effect on ovarian cancer cells.According to the evaluation of several strategies,the nano-hydrogel strategy combines the knowledge of pharmacokinetics and drug delivery theories,which can solve the existing problems of HDACIs to the greatest extent,and has a great application prospect in clinical application.