Synthesis And In Vitro Evaluation Of Tumor Micro-environment Degradable Poly(Doxorubicin) Based Prodrug

The design and synthesis of leakage-free drug delivery system?DDS?with high drug content has always been an essential challenge in the development of anti-cancer drug delivery system.The drug leakage-free design can effectively minimize the systematic toxicity of the drug delivery system,and a higher drug content will ensure the amount of the usage of the DDS can be reduced.The development of drug self-delivery system?DSDS?can be applied to fulfil these requirements.This paper aims at the design and preparation of a new drug leakage-free poly doxorubicin-based prodrug self-delivery system with high drug content and the details were discussed as following.1)A serial of doxorubicin?DOX?dimers could been prepared with different linkers.The D-DOX_carar and D-DOX_MAH could be prepared with the reaction of the amino group on DOX and with the linkage of carbamate bond or amide bond.The D-DOX_ADH was prepared with hydrozone bond and with the reaction of the carbonyl on DOX.These different DOX dimer prepared with different reaction site on DOX presented different physical and chemical properties.All these DOX dimers could be prepared into their corresponding prodrug nanoparticles by using nano-precipitation technology with proper good solvents and poor solvents.It was found that by adjusting the concentration of the DOX dimer in their good solvent during the nano-precipitation process,the size of the prodrug nanoparticles could be controlled.The DOX dimers could also be modified with polyethylene glycol?PEG?,and then the PEGylated DOX dimer prodrug nanoparticles could be prepared by the co-precipitation of DOX dimer with their PEGylated dimers.By adjusting the mass ratio of PEGylated DOX dimers to DOX dimers during the nano-precipitation process,prodrug nanoparticles with different particle size could be prepared,and hence,the drug content of the PEGylated prodrug nanoparticles could be precisely controlled.These different dimer nanoparticles showed different chemical reactivity and different decomposition behavior:D-DOX_ADH dimer based prodrug nanoparticles presented the highest solubility in DI and fastest drug release behavior;while the D-DOX_car dimer based nanoparticles shown the slowest decomposition rate.The preparation and the related research of DOX dimer DSDS discussed above laid a solid basis for the preparation of poly?doxorubicin?prodrug.2)By the composition of D-DOX_ADH and D-DOX_car dimers,the drug release rate tunable DOX dimer-based prodrug drug self-delivery system could be prepared.Comparing prodrug nanoparticles with different D-DOX_ADH and D-DOX_car mass ratios in their in vitro drug release experiments,(in this section,mixture with different ratios applied were named as D-DOX_mix-1,D-DOX_mix-3,D-DOX_mix-5,D-DOX_mix-3a and D-DOX_mix-3b),it could be found that,the drug release behavior of D-DOX_ADH molecular could be greatly affected by the addition of D-DOX_car which has a low drug release rate.In addition,in vitro cellular experiment shown that,after been cultured with the same concentration of different D-DOX_mix nanoparticles,the cell viability of the Hep G2 cancer cell decreased as the mass ratio of D-DOX_car increases.This research of the drug release rate-tunable DSDS provides a new approach to design more facile DDS.3)Based on the researches of DOX dimer molecules,pH and GSH dual responsive poly?doxorubicin?prodrug DSDS(PDOX_SS-NP)was prepared.With a two-step condensation process,the poly?doxorubicin?prodrug molecular can be prepared with 3,3'-dithiodipropionic acid and adipic dihydrazide with a high overall yield.The poly?doxorubicin?prodrug molecular had a number average molecular weight?M_n?of 1.66×10⁴ g/mol and a high drug content of 78%with DOX on the backbone of the polymer chain.The solubility of PDOX_SS was significantly reduced.The drug leakage rate of PDOX_SS-NP in the in vitro drug release experiment was only 0.60%over60 h at the simulating physiological medium?pH 7.4?,which could effectively reduce the toxicity to normal cells and tissues during the blood circulation.With the stimulation of acidic environment and high GSH concentration,the polymer chain of PDOX_SS-NP could be disintegrated and release DOX and DOX prodrug molecular with small molecular,which exhibits anti-cancer activity.PDOX_SS-NP could complete decompose within 36 hours when solid content is relatively low.In vitro cellular experiment shown that the PDOX_SS-NP could efficiently inhibit the cell proliferation of the Hep G2 cancer cell.After been cultured with PDOX_SS-NP at the concentration of 20?g/mL for 48 hours,the cell viability of Hep G2 cells was 54.8%.4)PEGylated poly?doxorubicin?prodrug DSDS(PDOX_MAH-PEG-NP)was prepared utilizing MAH and ADH as linker.The synthetic process of poly?doxorubicin?PDOX_MAH was firstly optimized and the PDOX_MAH-1 prodrug molecule with higher molecular weight and optimal drug release performance was obtained.Then,basing on the synthesis process of PDOX_MAH-1,the preparation of the PEGylated poly?doxorubicin?prodrug PDOX_MAH-PEG was achieved in a one step process utilizing D-DOX_ADH and DOX-ADH-DOX-PEG as monomer and MAH as linker.By tuning the molar ratio of D-DOX_ADH and DOX-ADH-DOX-PEG,PDOX_MAH-PEG_9:1,which had a higher M_n?3.1×10⁴ g/mol?and higher drug content?75.42%?,could be prepared.In the following in vitro drug release experiments,the PDOX_MAH-PEG_9:1-NP presented a low drug leakage with a drug releasement of 4.39%at pH 7.4 over 60 hours and with a cumulative drug release of 39.83%at pH 5.0,suggesting the effective pH responsive drug release behavior.It was also found that the PDOX_MAH-PEG_9:1-NP still presents a concentration related drug release behavior.The PDOX_MAH-PEG_9:1-NP could complete decomposed within 60 hours with a lower dosage.The PDOX_MAH-PEG_9:1-NP also shown good tumor cell viability inhibition ability in the in vitro cellular experiment.The cell viability of Hep G2 cells decreased to 55.23%after been cultured with PDOX_MAH-PEG_9:1-NP with a concentration of 20?g/mL for 48 hours.5)By modifying the chemical formulation of the poly?doxorubicin?prodrug molecule,P?Doxaz?-NP with enhanced anti-tumor efficiency could be prepared.Through a two-step reaction of DOX with hydrazine hydrate and formaldehyde,acid sensitive P?Doxaz?-NP with a M_n of2.1×10⁴ g/mol could be prepared with high yield.The P?Doxaz?-NP exhibited an even higher drug content than PDOX_SS-NP and PDOX_MAH-PEG9:1-NP prodrug DSDS,reached 87.92%.The main reason of the higher drug content was the optimization of the polymer structure by applying shorter intermolecular bridge fragments.The following in vitro drug release experiment shown that P?Doxaz?-NP could complete decomposition within 9.5 hours at pH 5.0 with a drug leakage of12.90%at pH 7.4 over 12 hours.The enhanced anti-cancer efficiency was then confirmed by the in vitro cellular experiments.After been cultured with 20?g/mL P?Doxaz?for 12 hours,the cell viability of Hep G2 cells was 54.83%,lowering than the free DOX which possessed a cell viability of 60.31%,which might result from the high drug content,fast decomposition and the stronger DNA binding ability of Doxaz produced during the decomposition process.These poly?doxorubicin?prodrug exhibited high drug content,and the good control over drug leakage or the enhanced anti-cancer efficiency could be achieved by optimizing the chemical structure.These poly?doxorubicin?based prodrug self-delivery system had a good potential in future cancer treatment.