Use Of 5-fluorouracil Loaded Micelles And Cisplatin In Thermosensitive Chitosan Hydrogel As An Efficient Therapy Against Colorectal Peritoneal Carcinomatosis

Objective: The aim of this study was to develop a novel hydrogel drug delivery system through the combination of 5-fluorouracil(5-FU)loaded polymeric micelles and cisplatin(DDP)in biodegradable thermosensitive chitosan(CS)hydrogel.Then,we established a colorectal peritoneal carcinomatosis(CRPC)mouse model to investigate the therapeutical effect of CS hydrogel drug system.Methods: Poly(-caprolactone)-poly(ethylene glycol)-poly(-caprolactone)(PCEC)copolymers with the designed molecular weight of 3700 was synthesized by ring-opening copolymerization of ?-CL and PEG(Mn = 2000)macromolecular initiator catalyzed by Sn(Oct)2.The 5-FU micelles were prepared by self-assembly of PCEC copolymers.Then,a dual drug delivery system was prepared through the combination of a cisplatin(DDP)-containing CS thermosensitive hydrogel and 5-FU micelles and was characterized in drug release behavior.The particle size of 5-FU micelles was evaluated using a dynamic light scattering(DLS,NanoBrook 90 Plus Zeta,Brookhaven,USA).Transmission electron microscope(TEM,Tecnai G2 F20,FEI,USA)was used to observe 5-FU micelles morphology.Drug loading(DL)and encapsulation efficiency(EE)of 5-FU micelles was determined by a high performance liquid chromatography(HPLC,Agilent,USA)instrument with a reverse phase C18 column(4.6 × 150 mm,3.5 ?m particle size).A modifieddialysis method was employed to determine the release behavior of 5-FU from free 5-FU and 5-FU from 5-FU micelles and independent release behavior of5-FU and DDP form the prepared CS hydrogel drug in vitro.The in vitro cytotoxicity of blank PCEC,FU and 5-FU micelles on CT26 cells were determined using standard MTT assay.The CS hydrogel drug thermosensitive characteristic was also measured by rheometer(TA Instruments,USA).BALB/c mice were used to establish the CRPC mice model,Tumor-bearing mice were randomly divided into the following six independent treatment groups(each group was 12,half of the mice were observed the survival time,the remained for immunohistochemistry):(1)normal saline(NS),(2)blank micelles-hydrogel,(3)5-FU micelles(20 mg/kg),(4)DDP loaded CS hydrogel(1 mg/kg),(5)FU(20 mg/kg)and DDP(1 mg/kg),(6)CS hydrogel drug.Next,mice were intraperitoneally treated with a single dose of the above mentioned compounds,200 ?L each compound.To evaluate the anti-tumor effect,mice general health status(6 mice per group)was monitored every day,and they were sacrificed on day 21.After day 21,tumor bearing mice in NS group began to die.The number of tumor nodules and tumor weight were measured.Tumor tissues were collected for immunohistochemical analysis.In addition,liver and lung tissues were collected for hematoxylin-eosin(H&E)staining to evaluate the presence of tumor metastasis.Expression of Ki-67 by immunology analysis was used to evaluate the mechanisms of anti-tumor.Results: The 5-FU micelle was obtained and achieved a high encapsulation efficiency.The average particlesize of the micelles was 22.56 nm,and it was mono-dispersed with a very narrow particle size distribution.Furthermore,the particle sizes of the micelles increased with increasing drug/polymer mass ratio,although a slight change was observed.5-FU and DDP release from the CS hydrogel drug exhibited no initial burst but showed a sustained release behaviors.The results of MTT demonstrated that the 5-FU micelle had superior cytotoxicity compared to FU,and that blank PCEC micelles did not possess noticeable cytotoxicity and could be used as safe drug delivery carriers.Both blank CS hydrogel and CS hydrogel drug exhibited an apparent thermosensitivity.The in vivo studies demonstrated that the mean number and weight of tumor nodules in the CS hydrogel drug group(10.33 ± 2.66,0.49 ± 0.11 g)were clearly decreased compared with those in the NS group(53.83 ± 9.99,2.31 ± 0.38 g,P <0.001),blank micelles-hydrogel group(52.67 ± 6.12,2.26 ± 0.28 g,P <0.001),5-FU micelles group(22.5 ± 4.23,0.99 ± 0.17 g,P < 0.001),DDP loaded CS hydrogel group(23.33 ± 3.56,0.98 ± 0.13 g,P < 0.001),and FU + DDP group(18.16 ± 3.06,0.79 ± 0.13 g,P <0.05).The finding indicated that CS hydrogel drug suppressed the growth of implanted tumor.And the blank micelles-hydrogel group did not show any anti-tumor activity.The median survival in the CS hydrogel drug group(43 days)was remarkably longer than NS group(25 days),blank micelles-hydrogel group(26 days),5-FU micelles group(31 days),DDP loaded CS hydrogel group(33 days),and FU + DDP group(35 days).The CS hydrogel drug system could also successfully inhibit the presence of metastaticlesions to the liver and lung tissues,as demonstrated by H&E staining.The Ki-67 positive cells in the CS hydrogel drug group(21.83 ± 5.71%)were significantly decreased compared with NS group(83.67 ±4.89%,P < 0.001),blank micelles-hydrogel group(80.67 ± 3.78%,P < 0.001),5-FU micelles group(57.00 ± 5.66%,P < 0.001),DDP loaded CS hydrogel group(55.50 ±4.64%,P < 0.001),and FU + DDP group(39.50 ± 3.94%,P < 0.001),respectively.Conclusion: Overall,our results showed several advantages of CS hydrogel drug system such as thermal-sensitivity,toxicity-free,and anti-tumor activity,suggesting that a safe and in situ gel-forming controlled drug delivery system for intraperitoneal application might represent the ideal system for a safer,effective and efficient chemotherapy against colorectal peritoneal carcinomatosis.The excellent properties of the system we created demonstrated that an in situ targeted therapy can be achieved.Accordingly,the pharmaceutical preparations may has a satisfactory clinical application perspective,which can be used for in situ treatment of tumor.