Preparation Of Monodispersed Dendritic Large-Pore Mesoporous Silica Nanoparticles Loaded With Therapeutic Agents Via A Dual-Templating Strategy

Objective:Dendritic large-pore mesoporous silica nanoparticles（DLMSNs）with center-radial pore structure is a new type of porous material.Compared with conventional mesoporous silicon nanoparticle（MSNs）,DLMSNs have attracted great interest due to their high surface areas with improved accessibility to the internal surface,controllable particle size,tunable pore size and pore volume,and easily realized dual functionalizations on both surface and internal of the particles.Two methods,the biphasic/emulsion approach and dual-templating method were mainly used to prepare the DLMSNs in the previous reports.The reported DLMSNs were mainly prepared with toxic organic solvents or auxiliary template agents,which were difficult to be cleaned in the post-treatment process,which would limit their biomedical applications.The large-scale green synthesis of DLMSNs with particle size less than 100 nm is extremely important for its applications in the biomedical field.Herein,taking therapeutic agents as auxiliary templates,we used one-step method to synthesis the therapeutic agents loaded DLMSNs with particle size less than 100 nm via a dual-templating strategy.Content:This study is divided into three parts.Firstly,we used the cationic surfactant cetyltrimethylammonium bromide（CTAB）as the main template and the negatively charged amphiphilic molecules under basic conditions（sodium salicylate（NaSal）,ferrocenecarboxylic acid（FCA）or hematoporphyrin dihydrochloride（HP））as auxiliary templating agents to prepare DLMSNs which are monodispersed and contain both microporous and mesoporous.Secondly,FCA was used as an auxiliary template to explore the formation mechanism of DLMSNs.The mechanism was also verified by using other electronegative amphiphilic molecules under alkaline conditions as auxiliary templates（ibuprofen（IBU）,deferasirox（DFS）,（sodium diethyldithiocarbamatetrihydrate）DDTC,methotrexate（MTX）,1,4,7,10-tetrazocyclododecane-1,4,7,10-tetraacetic acid（DOTA）and protoporphyrin disodium（NAPP））.Thirdly,the auxiliary templates would remain in DLMSNs after removing CTAB by choosing the appropriate treatment,which further played a therapeutic role in tumor therapy.Methods:1.Synthesis and characterization of DLMSNsDLMSNs were prepared via a one-step synthesis by using cationic surfactant CTAB and auxiliary template（NaSal,FCA or HP）as dual templates,tetraethyl orthosilicate（TEOS）as a silica source,and triethanolamine（TEA）as a catalyst and solubilizer for auxiliary templates in aqueous solution.Next,the nanoparticles obtained in this study were characterized.The morphology,particle size and dispersity of DLMSNs were characterized by transmission electron microscope（TEM）and scanning electron microscopy（SEM）.Particle size and polydispersity coefficient of nanoparticles in aqueous solution were determined by dynamic light scattering（DLS）.Adsorption-desorption isotherm and microscopic pore structure characteristics（specific surface area,pore volume and pore diameter distribution）of DLMSNs were characterized by N₂ adsorption desorption method.The obtained nanoparticles were characterized by fourier transform infrared spectrometer（FT-IR）to confirm that CTAB had been removed in DLMSNs.2.Exploration for the formation mechanism of DLMSNsWith a certain amount of FCA as the auxiliary template agent and CTAB as the main template agent,we changed the feeding strategies for CTAB and auxiliary templates.DLMSNs prepared by different methods were characterized by TEM to explore the assembly principle of the two templates.The influence on the formation of DLMSNs was investigated by changing the reaction temperature or reaction time.The mechanism was further verified with IBU,DFS,DDTC,MTX,DOTA or NAPP as auxiliary templates,and the morphologies and particle sizes of DLMSNs prepared under various reaction conditions were characterized by TEM.3.Application of DLMSNs in biomedicineIR780@DLMSNs was prepared by loading IR780 on DLMSNs prepared with NaSal as the auxiliary template,and their in vivo distribution was investigated by a in vivo fluorescence imaging system.The content of FCA or HP in DLMSNs was determined by inductively coupled plasma mass spectrometry（ICP-MS）or ultraviolet-visible-near infrared radiation spectroscopy（UV-Vis-NIR）after the removal of CTAB by selecting appropriate methods.The yield of reactive oxygen species（ROS）after the relevant DLMSNs being irradiated by X-ray or NIR laser were determined by using DCFH as single state oxygen fluorescence probe.Results:1.Synthesis and characterization of DLMSNsThe TEM images,SEM images and DLS results showed that the particle size and pore diameter of DLMSNs both increased with the amount of auxiliary template increasing.From the observation of TEM images,the particle sizes of DLMSNs ranged from 47 nm to 103 nm.The polydispersity indexes（PDIs）of these DLMSNs were all less than 0.14 in DLS analysis,which implied that the DLMSNs were dispersed well and physically stable in aqueous solution.The N₂adsorption-desorption isotherms of all DLMSNs samples demonstrated a H3 type hysteresis loop of the type IV isotherm in the relative pressure（P/P0）range of 0.4-1.0,suggesting the presence of different sized pores containing both micropores and mesopores.The pore size distribution map shows that the pore size range is 1.3-20 nm.All samples of DLMSNs have a large specific surface area（700-900 m²/g）.FT-IR spectrum showed that all DLMSNs after CTAB being removed have no characteristic peaks of methylene at about 2925 cm^-1,2854 cm^-1and 1473 cm^-1,indicating that the extraction process adopted has completely removed the surfactant CTAB.2.Exploration for the formation mechanism of DLMSNsIn the exploration for the formation mechanism of DLMSNs,three different feeding strategies for CTAB and auxiliary templates were adopted.In the first method,CTAB and FCA dissolved separately in aqueous solutions were added to the TEA aqueous solution simultaneously.The MSNs prepared by this way had a rambutan-like pore morphology.In the second method,CTAB and FCA in their solid states were added to the TEA aqueous solution simultaneously.The generated DLMSNs showed relatively large pore size,some largely sized particles,porous silica sheets,and random structured silica products due to disorderly assembly of FCA with CTAB.In the third method,CTAB were added in TEA aqueous solution firstly,and then FCA as auxiliary template agent was added after the micelles of CTAB were completely formed.The pore morphology of the generated DLMSNs had a regular stellate architecture,while the particles showed excellent monodispersity from TEM images.The mechanism is proposed as followed:The micelles self-assembled by CTAB are firstly formed in aqueous solution,and then the auxiliary templates are added in their solid states.In the course of dissolution,the molecules of these negatively charged auxiliary templates can close to the CTAB micelles via electrostatic interaction and further insert into these micelles using their hydrophobic domains in their molecules.Thus,the micelles co-assembled by two templates become swollen and direct the growth of silica-based walls among their blocks to induce the final dendritic pore network.The particle size and the wrinkle amounts of the DLMSNs increased with the reaction temperature increasing and the reaction time lasting.The center-radial mesoporous silicon nanoparticles could be prepared by using IBU,DFS,DDTC,MTX,DOTA or NAPP as auxiliary templates respectively,which further proved the mechanism of DLMSNs formation.3.Application of DLMSNs in biomedicineThe results of DLMSNs in vivo distribution showed that IR780@DLMSN nanoparticles could accumulate in tumor sites after injection through the tail vein,suggesting their good EPR effect.CTAB template were removed from DLMSNs by selecting different methods according to the chemical properties of FCA and HP and a certain amount of FCA and HP were retained in DLMSNs,respectively.A large amount of ROS could be generated after the relevant DLMSNs being irradiated by X-ray or NIR laser,respectively.Conclusion:In this study,DLMSNs with tunable pore size,pore diameter,pore volume and pore morphology were successfully prepared via a dual templating strategy by using the therapeutic agents as an auxiliary template and the aqueous solution as a single reaction medium.The obtained DLMSNs were uniform and smaller than 110 nm.The results of TEM,SEM and DLS showed that DLMSNs had excellent monodispersity.We further explored the formation mechanism of DLMSNs based on the co-assembly of CTAB and auxiliary templates.We derived that an auxiliary template with amphiphilic structure and electronegativity under alkaline conditions was suitable for co-assembling with CTAB micelles and promoted the formation of DLMSNs.We predicted a series of therapeutic agents or imaging agents as auxiliary templates and used them to prepare DLMSNs with different pore sizes,pore volumes and pore morphology.By selecting appropriate methods to remove the template agent CTAB,a certain amount of therapeutic agents FCA or HP was still retained,and exerted potential anti-tumor effects.This study provided a simple and controllable way to prepare DLMSNs with controllable particle size,pore size,pore volume,pore morphology and therapeutic agents,which is promising as a nanoplatform for biomedical applications.