| Objective In order to improve the effect of photodynamic therapy in the treatment of pancreatic cancer, this study regards the nanoparticles NP-PPE/Ce6 which encapsulated the photosensitizer of Chlorin e6 (Ce6) as photo sens it izer delivery system, exploring the improvement of nanoparticles NP-PPE/Ce6-mediated photodynamic therapy in the treatment of pancreatic cancer, so as to provide a new therapeutic choice for the effective treatment of pancreatic cancer in clinic.Methods The nanopartticles NP-PPE/Ce6 were prepared by self-assembly of amphiphilic polymers with photosensitizers Ce6 in aqueous solution.We detected the size distribution and stability of NP-PPE/Ce6 by using Dynamic Light Scattering. The morphology of the particles NP-PPE/Ce6 was observed by Transmission Electron Microscope. And the release behavior of NP-PPE/Ce6 was measured by fluorescence and HPLC, respectively. The ability of ROS generation of NP-PPE/Ce6 in the cellular level and the extracellular lever was determined by fluorospectro photometer with the help of dichlorofiuorescein diacetate (DCFH-DA) as an indicator. The cellular uptake of NP-PPE/Ce6 to BxPC-3 cells was monitored by Flow Cytometry or Laser Scanning Confocal Microscopy. Cytotoxicity of NP-PPE/Ce6 on pancreatic cancer cells was detected by MTT cytotoxicity assay. Using trypan blue solution stain monitor the pancreatic cancer cell apoptosis. To detect the blood cleared and tissue distribution and enrichment of nanoparticles NP-PPE/Ce6, the HPLC and imaged with a Xenogen IVIS Lumina system were used. Finally, in order to verify the inhibition effect of NP-PPE/Ce6 for pancreatic cancer BxPC-3, the change of tumor volume after photodynamic treatment were observed. And after treatment, the mice were sacrificed and the tumor sections were acquired for immunnohistochemical staining of the terminal transferase dUTP nick-end labeling (TUNEL) assay and the proliferating cell nuclear antigen (PCNA).Results 1. The cellular lever study of NP-PPE/Ce6 combination with photodynamic therapy for pancreatic cancer:(1)firstly, we successfully prepared the Ce6-loaded nanoparticles NP-PPE/Ce6, and its physical and chemical characteristics were tested. The results shown that NP-PPE/Ce6 were a spherical particles and its size was 44.4±4.3nm, the particles size distribution (PDI) was 0.108. And the study indicated that NP-PPE/Ce6 had a good stability. It also significantly accelerated the release rate of photosensitisers Ce6 in an acidic environment of pH 5.5. Then the capacity of producing active oxygen after laser irradiation by detecting the DCF fluorescence signal. (2)At the cellular level, we found that NP-PPE/Ce6 could promote the cellular uptake of photosensitizers Ce6 for BxPC-3 cells and after laser irradiation, compared to free Ce6, with the NP-PPE/Ce6 treated the active oxygen was significantly increased in BxPC-3 cells. By the MTT assay both free Ce6 and NP-PPE/Ce6 showed negligible cytotoxicity without irradiation. After irradiation with a laser, the cell viability decreased in a dose-dependent manner for free Ce6. However, NP-PPE/Ce6 was more effectively reduced cell viability than the free Ce6. Moreover, the apoptosis cells were further analyzed by trypan blue staining after NP-PPE/Ce6 incubation and NIR-laser treatment. In accordance with the results in the MTT assay, a large number of apoptotic cells dyed blue were observed for NP-PPE/Ce6 group, while fewer apoptotic cells were detected in the free Ce6 group. In addition, the groups without laser irradiation did not show any apoptosis incubated with free Ce6 or NP-PPE/Ce6 at the same conditions. They indicated that NP-PPE/Ce6 improved the effects of photodynamic therapy to pancreatic cancer cells.2.The study of NP-PPE/Ce6 combination with photodynamic therapy for pancreatic cancer in vivo, the tumor growth was significantly inhibited after treatment with NP-PPE/Ce6 plus irradiation, the volume of the tumor even reduced about 60% compare to the original size. However, treatment with free Ce6 plus NIR laser irradiation resulted in slight inhibition of tumor growth. In addition, administration of NP-PPE/Ce6 without NIR irradiation did not inhibit BxPC-3 tumor growth in comparison to PBS, indicated that the photosensitizer Ce6 did not exhibit phototoxicity without laser stimulation. In addition, the tumor was excised after the last measurement, and the weight of the tumor mass was assessed. The mean tumor weight treated with NP-PPE/Ce6 without laser irradiation or with Ce6 plus laser irradiation was 3-fold and 2-fold of that treated with NP-PPE/Ce6 plus laser irradiation. Compared to other groups, the PCNA and TUNEL signals of NP-PPE/Ce6 with laser group were significantly enhanced.Conclusion With the delivery by NP-PPE/Ce6, nanoparticles can effectively prolong the time of blood circulation, increase tumor concentration and can effectively reduce the side effects produced by the non-targeted. Administration of NP-PPE/Ce6 plus NIR irradiation markedly enhanced the inhibiting of BxPC-3 tumor growth, indicating the great potentials of polyphosphoester-based nanocarrier as the delivery system of photosensitizer for PDT of pancreatic cancer. |
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