Chimeric Peptide Nanorods For Plasma Membrane And Nuclear Targeted Photosensitizer Delivery And Enhanced Photodynamic Therapy

Photodynamic therapy(PDT)is a new method for the treatment of tumor diseases with photosensitive drugs and laser activation.Using a laser with specific wavelength to illuminate the tumor site can activate the photosensitive drug,so that the drug can selectively accumulate in the tumor tissue and trigger the photochemical reaction to destroy the tumor.PDT consists of photosensitizer(PS),molecular oxygen and laser.In PDT treatment,a photosensitizer is first injected into the patient’s body or covered on the skin surface for a certain period to internalize it into the tissue.Then the target tissue is irradiated by a prepared excitation light with appropriate wavelength,which forms reactive oxygen species(ROS).Reactive oxygen species can destroy cancer cells via apoptosis-mediated cell damage,blood vessel injury and immune response activation.Compared with traditional therapies,there are some advantages of PDT with double selectivity,no drug resistance,non-invasive treatment,and precise treatment with low side effects.However,it can’t be ignored that the laser irradiation will cause toxicity in the non-targeted part with PS,which is why drug targeting is currently popular.The drugs release not only in the target tissues but also in other organs and tissues,which causes toxic and side effects on non-targeted tissues.At this time,photosensitizers that can directly target cancer cells and even organelles are particularly important.The cell membrane,as a barrier,prevents extracellular materials from entering into the cell freely to ensure the relative stability of intracellular environment and various biochemical reactions in an orderly manner.The nucleus is the regulatory center of cell heredity and metabolism,playing an important role in a eukaryotic cell.Compared with the single target(such as mitochondrial and lysosome)reported in other reports,the dual targets of cell membrane and nucleus can disrupt cell defenses and promote drug endocytosis,which more precisely acts on the nucleus to prevent cell replication and division,thereby controlling the development of cancer.This provides insight into the design of a self-delivery chimeric peptide nanorod for dual targets of the membrane and nucleus,which can directly reach the destination to greatly reduce its toxic side effects and realize precise treatment.Objective:In this work,we rationally fabricated the self-delivery chimeric peptide nanorods(denoted as pnPNP)for plasma membrane and nuclear targeted delivery of photosensitizer to achieve a coordinated in-situ photodynamic therapy and further observed its application in tumor precision treatment.To highlight the advantages of pnPNP dual-targeting PDT,pnNP with nuclear targeting was designed for the control group.Methods:1.Characterizations of pnPNP and pnNP:The particle size and zeta potential were characterized by Nano-ZS ZEN360.The morphology was observed by TEM.The UV-vis absorbance was measured by UV-vis spectrophotometer.2.ROS production ability of pnPNP and pnNP:The production of ROS was measured by fluorescence spectrometer and CLSM using SOSG and DCFH-DA as the indicators,respectively.3.Cellular uptake and light-triggered lysosomal escape:CLSM was employed to investigate cellular uptake and light-triggered lysosomal escape.4.Plasma membrane anchoring and nuclear targeting ability of pnPNP and pnNP:CLSM was employed to investigate the plasma membrane anchoring and nuclear targeting ability of pnPNP and pnNP to He La,CT26 and 4T1 cells.5.In vitro cytotoxicity measurements:The cytotoxicities of pnPNP and pnNP were measured by MTT,flow cytometry and CLSM.6.Anti-tumor studies:4T1 tumor-bearing mice were employed to evaluate the anti-tumor effects of pnPNP when the tumor volume reached about 100 mm~3.The mice were randomly divided into five groups(5 mice in each group),including PBS,pnPNP,pnPNP with irradiation,pnNP and pnNP with irradiation.pnPNP and pnNP were intravenously injected into the tumor bearing mice respectively.For PDT,the mice were subjected to the laser irradiation for 5 min at 20 h after administration.The tumor volume and the body weight were monitored every other day.After 15 days,the tumor tissues were obtained for photographing and weighting.Results:1.Dynamic light scattering(DLS)measurement revealed the well-dispersed nanoparticles of pnPNP and pnNP with narrow size distribution.The TEM image showed that the nanorods had an aspect ratio of about 3 and had a positive charge,which was beneficial for nuclear penetration.It could be seen from the UV spectrum that pnPNP and pnNP had sharp peaks at about 400 nm,and there were significant Pp IX characteristic absorption peaks at 500-700 nm.2.SOSG showed that few of fluorescence changes were found in pnPNP and pnNP groups when without light irradiation,while under light irradiation,a faster fluorescence enhancement was found in pnPNP group rather than in pnNP group.Similar results were also found in the intracellular ROS detection(DCFH-DA)by CLSM.The strongest green fluorescence was detected in pnPNP group with light.3.The uptake of both pnPNP and pnNP by 4T1 cells enhanced with the increase of incubation time.But the fluorescence intensity of 4T1 cells treated with pnPNP was about 10-fold higher than that of cells treated with pnNP.The Lyso-Tracker Green was found to be a good match with pnPNP and pnNP,indicating that the endocytic pathway of the nanorods was lysosome.After light exposure,the green fluorescence signal disappeared in 4T1 cells,which might be attributed to the efficient photodynamic destruction of the lysosomal structure by pnPNP and pnNP.4.Whether in 4T1,He La or CT26,the red fluorescence of pnPNP overlaps with the green fluorescence of cell membrane dye Di O,which proved the excellent plasma membrane targeting ability of pnPNP.In the absence or presence of light irradiation,the nuclear targeting ability of pnPNP and pnNP could be observed.5.In vitro cytotoxicity showed that both pnPNP and pnNP exhibited a low dark toxicity and concentration-dependent photo toxicity against 4T1 and He La cells,and compared with pnNP,pnPNP was observed to have significantly higher phototoxicity.6.In vivo anti-tumor studies had shown that,just like PBS,injection with pnPNP or pnNP without light could scarcely inhibit the tumor growth.Once exposed to the light,both pnPNP and pnNP had a signifificant suppression to the tumor growth,but pnPNP was significantly better than pnNP(P<0.05).Compared with single-target pnNP for photodynamic tumor therapy,dual-target pnPNP had huge advantages.Conclusion:In conclusion,the self-delivery chimeric peptide nanorods(denoted as pnPNP)were developed for plasma membrane and nucleus dual-targeted photosensitizer delivery to achieve the synergetic in situ PDT.In vitro studies had shown that the dual targeting ability was beneficial to the efficient subcellular localization of pnPNP in plasma membrane and nucleus.Plasma membrane targeted PDT of pnPNP could improve the membrane permeability to enhance the cellular uptake,even trigger the membrane disruption and cause cell necrosis directly.Upon light irradiation,the internalized pnPNP could effectively disrupt lysosomal structures and realize the nuclear permeation for supplementary nuclear targeted PDT.Abundant experiments verified that the plasma membrane and nucleus dual-targeted PDT exhibited a far better anti-tumor effect than the single nuclear targeted PDT.This synergistic subcellular dual-targeting strategy maximized the PDT therapeutic efficacy,which also provided a new insight for the development of advanced drug delivery systems.