PH-sensitive Poly(Ortho Ester Urethanes)Drug Carriers:Synthesis,Characterization,in Vitro And In Vivo Antitumor Evaluation

Biodegradable polymeric nanocarriers have been extensively explored for antitumor drug delivery because of their high drug loading efficiency and encapsulation efficiency,ability to optimize the distribution of drugs in vivo and reduce the side effect of drugs,prolonged drug circulation time.Nowadays,researchers have explored a series of polymer materials for drug delivery.Among these biomaterials,biodegradable polyurethanes as one type of synthetic biocompatible materials have been extensively investigated for various biomedical applications such as controlled drug release and tissue engineering because of their simple and efficient preparation method,outstanding biomedical properties.The biodegradability of polyurethanes was typically acquired by introducing degradable polyesters and polycarbonates into the polymer backbone.However,the degradation rate of these polyurethanes is generally slow,which often cannot meet the requirements of controlled drug delivery applications.In order to improve the rate of the degradation of these polyurethanes,researchers have introduced several stimuli-sensitive bonds such as acid-labile ketal,acetal,hydrazone bonds and reductively cleavable disulfide bonds into the backbone of polyurethanes.Ortho ester bonds as one family of acid-labile bonds have been explored for antitumor drug delivery.However,to the best of our knowledge,there is no report on the development of ortho ester-based polyurethanes and corresponding nanoparticles for antitumor drug delivery.In this paper,one type of pH-sensitive poly(ortho ester urethanes)(POEU1 and POEU2)were synthesized via polycondensation reaction of an ortho ester diamine with active esters of different polycaprolactone(PCL)diols(Fw=530 and 2000)under the mild conditions.POEU1 and POEU2 were selected for the fabrication of nanoparticles(NP1 and NP2)using an oil-in-water single emulsion technique.The structure and molecular weight of POEU1 and POEU2 were examined by 1H NMR and GPC,respectively.The results showed that POEU1 and POEU2 were successfully synthesized with number molecular weight of 2.2 and 4.09 × 104,respectively.Transmission electron microscopy(TEM)and dynamic light scattering(DLS)results revealed that the size of NP1 and NP2 is about 200 nm and the morphology of NP1 and NP2 is spherical-like.In vitro degradation studies showed that both of NP1 and NP2 were stable in the physiological condition(pH 7.4)and displayed a time-dependent degradation and decrease in size at pH 5.0.In addition,at pH 5.0,the rate of the degradaion of NP1 and NP2 could be regulated by the hydrophobicity of polymers.After 192 h,the ortho esters of NP1 prepared from less hydrophobic POEU1 was hydrolyzed completely,whereas those of NP2 prepared from more hydrophobic POEU2 was hydrolyzed by 83.41%?The nanoparticles(NP1 and NP2)prepared by POEU1 and POEU2 could encapsulate antitumor drug Doxorubicin(DOX)with high drug loading efficiency,and the obtained DOX-loaded nanoparticles were named as NP1/DOX and NP2/DOX,respectively.In vitro drug release studies showed that the release of DOX from NP1/DOX and NP2/DOX showed a significantly acid-accelerated behavior,and could be tuned by hydrophobicity of the starting polymers.At pH 5.0,the drug release rate of NP2/DOX prepared from more hydrophobic POEU2 was slower than that of NP1/DOX prepared from less hydrophobic POEU1.Confocal laser scanning microscopy(CLSM)and flow cytometry were used to evaluate the qualitatively and quantitatively cellular uptake of NP1/DOX and NP2/DOX towards human neuroblastom cancer cell line(SH-SY5Y)and murine hepatic cancer cell line(H22),respectively.The results demonstrated that NP1/DOX and NP2/DOX could be readily internalized by SH-SY5Y cells and H22 cells,and then DOX rapidly moved into nucleus.MTT measurements were used to examine the cell viability of SH-SY5Y and H22 cells which were incubated with blank nnanoparticles(NP1 and NP2)and DOX-loaded nanoparticles(NP1/DOX and NP2/DOX)for 24 h,respectively.The results showed that the viability of SH-SY5Y cells and H22 cells incubated with NP1 and NP2 were over 95%even up to the highest concentration of 1 mg/mL,demonstrating excellent biocompatibility of the blank nanoparticles.However,NP1/DOX and NP2/DOX could efficiently kill SH-SY5Y cells and H22 cells and displayed a dose-dependent decrease in cell viability.A three-dimensional(3D)multicellular tumor spheroids prepar,ed from SH-SY5Y cells(3D SH-SY5Y MCTS),as in vitro tumor model,was used to further evaluate the antitumor effect of NP1/DOX and NP2/DOX.The results demonstrated that as compared to free DOX,NP1/DOX and NP2/DOX showed a significantly enhanced penetration and growth inhibition in 3D multicellular tumor spheroids(MCTS).H22 tumor-bearing mice were used to evaluating in vivo distribution and antitumor effect of DOX-loaded nanoparticles(NP1/DOX and NP2/DOX).In vivo drug distribution results showed that both of NP1/DOX and NP2/DOX showed a prolonged blood circulation time,improved accumulation in solid tumor and reduced cardiac toxicity of DOX,compared to free DOX.In vivo antitumor results indicated that,during the 7 days of treatments,the saline groups and blank nanoparticles(NP1 and NP2)groups displayed a continuing increase in volume of tumor,which was almost 8-9 folds larger than the staring volume after 7 days.However,free DOX and both of DOX-loaded nanoparticles(NP1/DOX and NP2/DOX)groups showed a significantly growth inhibition in tumor,and the inhibition rate(72.68%and 70.37%)of NP1/DOX and NP2/DOX was higher than that of free DOX(62.61%).Histological analysis results indicated that all DOX formulations-treated groups displayed a dramatically decline in tumor cell density band various degree of tissue necrosis,as compared to saline group.The necrosis regions in the DOX-loaded nanoparticles-treated groups were slightly larger than that of free DOX-treated group.A serious pathological damage and necrosis of the muscle fibers were displayed in cardiac tissues of free DOX-treated group,which indicated that free DOX had serious cardiac toxicity.Instead,no obviously pathological damage was observed in heart treated with DOX-loaded nanoparticles.In summary,such biocompatible and pH-sensitive poly(ortho ester urethanes)have enormous potential in drug delivery system for cancer therapy.