| Photodynamic therapy(PDT)has become a research hotspot in the field of tumor therapy due to its advantages of non-invasive and controllable time and space.Compared with traditional tumor treatment methods,photodynamic therapy also has the characteristics of low toxicity,good prognosis,strong universality,and high temporal and spatial selectivity.Studies have found that photodynamic therapy can induce immunogenic cell death(ICD)in tumors.While killing tumor cells,it releases DAMPs,mainly including calreticulin(CRT),adenosine triphosphate(ATP)and high-mobility group box 1 protein(HMGB1).Photosensitizers,lasers with specific wavelengths and oxygen are the three important elements for photodynamic therapy.Under the action of a certain dose of photosensitizer,light parameters(such as time,light intensity,wavelength,etc.)are the key factors affecting the efficacy of PDT.Excessive light time can cause skin ulceration and other physiological damages,while insufficient light time cannot achieve good therapeutic effects.It usually requires trial and error to screen out the best light parameters for PDT to make photodynamic therapy safer and more effective.As a microenvironmental parameter,intracellular viscosity plays a vital role in the signal transmission and interaction between biomolecules.When cancer cells undergo apoptosis,the intracellular viscosity will increase significantly.There are few studies on the changes of intracellular viscosity when cells undergo necrosis.However,when the cells undergo necrosis,the increased permeability of the cell membrane causes cell swelling,and finally leads to cell rupture.It is speculated that this process may cause a certain change in viscosity.Therefore,in order to better screen the light conditions of photodynamic therapy,it is of great significance to use the"molecular rotor",that is,the ratio fluorescent viscosity probe,to measure the viscosity change of the microenvironment in living cells in real time and accurately.In order to screen out the appropriate illumination parameters,this paper co-encapsulated the two-photon viscosity probe DSF and the photosensitizer PPa and prepared it into polymer micelles--DLM micelles,so that it can monitor the intracellular viscosity in real time during photodynamic therapy to screen out the best PDT conditions to induce ICD.The main research content of this article is divided into the following three parts:The first part is the preparation and characterization of DLM micelles.The DLM micelles were prepared by thin film hydration method,and the encapsulation efficiency of DSF and PPa in DLM micelles was 53.34±6.34%and 64.87±5.83%,and the drug loading was 1.49±0.35%and 2.13±0.56%,respectively;the particle size and Zeta potential of DLM micelles were 20.30±0.15 nm and-35.70±1.84 m V,respectively.The prepared DLM micelles are quasi-spherical and have a relatively uniform distribution.The storage stability of DLM micelles at 4℃and 37℃and the stability in serum were investigated.The results of stability showed that the DLM micelles remained relatively stable when stored at 4℃for 7 days,37℃for 3 days and 37℃for48 hours in serum.The fluorescence intensity and viscosity of DSF were measured.The results show that DSF,as a ratio fluorescent probe,can realize real-time monitoring of the viscosity of the intracellular microenvironment.The second part is the investigation of the effectiveness of DLM micelles in vitro.The MTT results showed that there was no obvious cytotoxicity of DLM micelles,and the cell survival rate decreased with the increase of light time and light intensity.Two-photon confocal microscope was used to monitor the fluorescence changes of DSF in cells and induce cell death in real time after laser irradiation:DSF fluorescence showed a trend of increasing at first and then decreasing with the prolonged light time,indicating that the intracellular viscosity has changed to a certain extent during laser irradiation.The dynamic process of cell membrane rupture is captured by marking the cell membrane,and it was observed that the cells swelled,deformed and ruptured sequentially during the light process.Through the analysis of ATP,HMGB1,CRT showed that PDT can not only kill tumor cells efficiently,but also induce immunogenic cell death and increase the release of immunogenic signal molecules.When the light intensity indicated by DSF is reached,the release amount did not increase significantly with the increase of light intensity.The third part is in vivo pharmacodynamic evaluation of DLM micelles.In the unilateral BALB/c mouse breast cancer model,the small animal in vivo imager was used to evaluate the targeting of DLM micelles.The results showed that the prepared DLM micelles can achieve passive targeting of tumor tissue.DSF could monitor the changes in intracellular viscosity during photodynamic therapy in real time in vivo.In the BALB/c mouse breast cancer bilateral model,the tumor immune response induced by photodynamic therapy was evaluated using the changes in the concentration of IFN-γ,IL-12,and TNF-αin the mouse serum as indicators.The results showed that the intravenous injection of DLM micelles through photodynamic therapy can increase the levels of IFN-γ,IL-12,and TNF-αcytokines in the serum of mice.Tumor volume growth,tumor inhibition rate,body weight,intratumoral CRT exposure,the maturation induction of dendritic cells and the activation of immune effector cells were analyzed.The results showed that within the light time range indicated by DSF,intravenous injection of DLM micelles through photodynamic therapy can enhance the exposure of CRT on the surface of tumor cells,stimulate the maturation of dendritic cells,thereby increasing the proportion of activated effector cells(NK cells,CD4~+T cells,CD8~+T cells)in the tumor,which not only effectively eliminates the primary tumor,but also stimulates the body to produce an immune response to the anti-tumor system,has a significant inhibitory effect on distant tumors,and has a certain inhibitory effect on liver and lung metastasis.With the continuous increase of light time,the therapeutic effect and the degree of inducing ICD did not continue to increase significantly,and prolonged photodynamic therapy treatment could significantly reduce the weight of tumor-bearing mice,causing skin redness,inflammation,and even ulceration.In summary,this paper uses the thin film hydration method to prepare DLM micelles loaded with the two-photon viscosity probe DSF and the photosensitizer PPa,and uses the"molecular rotor"feature of DSF to monitor the intracellular viscosity of tumor cells in real time to screen the best optimal PDT illumination parameters that induce immunogenic cells death,thereby activating or enhancing the anti-tumor immune response to enhance the therapeutic effect on breast cancer. |
