Responsive Dendrimer Vector For The Treatment Of Digestive System Tumors

The First Part Degradable Dendrimer Modified by p H and Reduction Step by Step Response for Gene Therapy of Hepatocellular CarcinomaBackground Micro RNA(mi RNA)therapy provides an alternative for the treatment of advanced malignant tumors.However,its therapeutic effect is usually hampered by mi RNA instability and off-target effect.Micro RNA-122(mi R-122),rich in normal liver tissue,has been shown to be significantly down-regulated in liver cancer,promoting hepatic carcinogenesis and tumor growth.One strategy is to deliver mi R-122 directly into cells to suppress tumor growth.However,mi R-122 treatment of liver cancer requires precise release at the tumor sites to reduce off-target effect and increase the accumulation of mi R-122 in the tumor.Dendrimers have attracted extensive attention because of the rich positive charge on the surface.But the paradox between low cytotoxicity and high transfection efficacy and excessive high positive charge leading to mononuclear phagocyte system(MPS)clearance undermine the applications of dendrimers.Functionalized dendrimers with programmable response to tumor microenvironment stimuli can promote the stability of mi R-122 in blood circulation,increase enrichment at the tumor site,thereby enhancing the efficacy of liver cancer treatment.Aim The purpose of this study was to synthesize modified degradable dendrimer polymer responsive to p H and reduction stimuli for gene therapy of liver cancer.Materials and Methods The GSSG was prepared by reacting a 3,3'-dithiodipropionate di(N-hydroxysuccinimide ester)(DSP)containing a disulfide bond with a second generation PAMAM(G2)at a molar ratio of 1:1.The GSSG was then reacted with PEG(MW = 5000 Da)containing a p H-responsive Dlinkm at a G2:PEG molar ratio of 1:1 to form the target compound BOMB.The compounds were characterized by 1H NMR and Fourier transform infrared spectroscopy(FT-IR).The gel electrophoresis experiments confirmed the nucleic acid loading capacity.Dynamic light scattering(DLS)and transmission electron microscopy(TEM)were utilized to examine the particle size and morphology of the BOMB system.The tumor microenvironment was simulated in vitro to study the nucleic acid release profile of the BOMB system.The human liver cancer Huh-7 cells were used as a model to verify the phagocytic capacity of the BOMB system in vitro.The expression level of mi R-122 in liver cancer treated by the BOMB/mi R-122 system was quantified by RT-q PCR and western blot assay to examine the RNA and protein expression,respectively.At the same time,we used Huh-7 cell-bearing nude mice as a model to verify the tumor suppression ability of the BOMB system.Results We successfully synthesized BOMB polymer confirmed by NMR and IR.The experiment results also showed that BOMB has favorable ability to condense mi R-122.The resulting BOMB/mi R-122 particles were spherical with diameters around 200 nm.BOMB/mi R-122,at lower p H simulating the micro-environment of liver cancer,the particle size of the nanoparticle decreased and the charge elevated,benefiting the internalization by liver cancer cells.Superior gene transfection efficiency was demonstrated by in vitro assays.When the low p H stimulation and reduction stimulation were asserted to the systems in a stepwise manner,the release of mi R-122 showed a significant stimulation dependence,and a rapid release of mi R-122 was observed in the presence of both p H stimulation and reduction stimulation.The results of in vivo fluorescence showed that the BOMB/mi R-122 system not only has long-circulation ability,but also has good tumor targeting ability.Compared with the naked mi R-122 gene therapy,the efficiency of BOMB/mi R-122 in inhibiting tumors was greatly enhanced.Immunohistochemistry and western blotting experiments also showed that the mi R-122 target protein of the liver cancer cells in the BOMB/mi R-122 treatment group was greatly down-regulated.Conclusion The BOMB system with programmable response to tumor microenvironment is simple and exquisite in synthesis.Mi R-122 can be targeted and released at the tumor site with controllability,thereby enhancing the precise therapeutic effect of liver cancer,indicating great application potential.The Second Part Reduction Response PEG Modified Novel Dendrimer Mediated Molecular Targeting Therapy for Pancreatic Cancer and Overcoming Drug ResistanceBackground Pancreatic cancer is a malignant tumor with a high mortality rate.Traditional chemotherapy has a poor effect on it.Some studies have found that m TOR inhibitors can effectively inhibit the occurrence and growth of pancreatic cancer.OSI-027 can simultaneously inhibit m TORC1 and m TORC2 complexes of m TOR,providing a new choice for the treatment of pancreatic cancer.However,OSI-027,like most molecular targeted drugs,suffers from:(1)poor water solubility leading to ineffective treatment concentration;(2)inadequate accumulation at the tumor site;(3)and problems such as drug resistance during treatment.Dendrimer carriers have hydrophobic cavities and hydrophilic shells,which are often used to carry hydrophobic drugs.After reductive response modification,OSI-027 can be specifically cleaved in the reductive environment of cancer cells.It can not only improve drug solubility and drug delivery mode,but also improve the tumor targeting of OSI-027.In addition,the introduction of nanomaterials has the potential to overcome drug resistance.Aim This study intends to develop a novel reduction-responsive modified dendritic nanocarrier for improving the water solubility of OSI-027 and targetability to tumor sites,promoting the therapeutic effect of molecular targeted drugs in pancreatic cancer and drug-resistant pancreatic cancer.Materials and Methods Herein,an eight-arm PEG-embedded dendrimer with reduction responsive disulfide bond was used to couple a commercial dendrimer PAMAM [second generation(p G2),three generations(p G3)] and a laboratory-derived dendrimer dendrimer [second generation(m G2),third generations(m G3)] to construct a series of reductively modified dendrimer polymers.The products were characterized by 1H NMR and the drug loading efficiency was detected by UV spectrophotometer.The interaction force between the material and OSI-027 was simulated by ATUO DOCK software.The particle size and morphology were examined by DLS and TEM.Drug resistant cells were induced by drug induction,and the cellular internalization behavior of the drug-loaded system was inspected by flow cytometry and laser confocal microscopy.For in vivo experiments,normal cell-bearing mice and drug-resistant cell-bearing mice were constructed.The fluorescence intensity of the materials carrying Cy5 was monitored by in vivo fluorescence to evaluate the tumor targeting ability of the material.The tumor size was measured periodically after administration.Furthermore,we explored the mechanism by which drug-loaded systems overcome drug resistance via transcriptome sequencing analysis.Results Our results indicated that the solubility of the water-insoluble targeted drug "OSI-027" was enhanced after the introduction of the MAP-m G3 delivery system,and the first-pass effect could be circumvented by intravenous administration.In vivo fluorescence experiments showed favorable enrichment effect at the tumor site,and the introduction of the vector confirmed the enhanced phagocytosis of the drug by measuring the intracellular drug concentration.In vitro experiments showed that MAP-m G3/OSI-027 exhibit superior tumor suppression both in normal cell tumor-bearing model and drug-resistant pancreatic cancer cell-bearing model.In vivo experiments further confirmed that MAP-m G3/OSI-027 had excellent anti-tumor effect,even if the dose was reduced by 50%.In addition,intravenous administration exerted better therapeutic effects than oral administration.Transcriptome sequencing revealed that the drug resistance gene ABCB1 was the main cause for cell resistance.Conclusion In summary,MAP-m G3 can greatly enhance the water-solubility of OSI-027 for intravenous administration.The drug delivery system of MAP-m G3/OSI-027 has a good anti-tumor effect in vitro and in vivo,and can overcome the drug resistance of pancreatic cancer.It can be further engineered into a potentially safe and effective delivery system for future biomedical and preclinical applications of other targeted therapies for malignancies.