| Malignant tumor is one of the most dangerous disease to human health.In recent years,in addition to chemotherapy,more and more new treatment strategies have been used in the treatment of cancer,such as photodynamic therapy(PDT).PDT involves stimulating photosensitizers at specific sites to produce reactive oxygen species(ROS)to kill tumor cells.However,most photosensitizers(PSs)have poor water solubility and poor tumor targeting.Traditional delivery of PSs by polymer encapsulation often has low drug loading,and may cause some toxicities of carrier-related materials,and even premature drug leakage.Compared with the polymer encapsulation method,the carrier-free pure drug self-assembled nanosystem has shown evident advantages in greatly improving the drug load,reducing the toxicity associated with excipients,and a simple preparation process with better reproducibility.However,carrier-free nanomedicines also have some difficulties to be solved,especially the poor stability.PPa is a porphyrin photosensitizer of chlorophyll family.Althoug the poor solubility,PPa has a strong conjugate structure.It is the first time that PPa was found to self-assemble into uniform nanoparticles(NPs)in aqueous solution.Furthermore,we designed a stable core-shell matched PPa/PPa-PEG2K NPs using the synthetic amphiphilic PPa-PEG2K polymers as stabilizing modifiers.Meanwhile,the PCL-PEG2K polymers with similar molecular weight were selected as the control group to prepare PPa/PCL-PEG2K NPs.Through molecular docking experiments,it was found that there were muitiple of forces between PPa molecules.Then,through the investigation of intermolecular destructive agents and the scanning of ultraviolet absorption and fluorescence spectrum,it was found that there were muitiple forces involved in the process of self-assembly to form nanoparticles,among which the hydrophobic action and ?-? stacking played a maj or role.The release profile of PPa/PPa-PEG2K NPs was investigated using phosphate buffer saline(PBS,pH 7.4)containing 20%tetrahydrofuran as the release medium.PPa solution showed a certain burst release phenomenon,while the NPs showed a certain continuous release characteristis.Compared with PPa/PCL-PEG2K NPs,the release of PPa/PPa-PEG2K NPs was slower.The result might be attributed to the core-shell matched modification,which made the NPs more stable and delayed the release of NPs.The cell uptake,intracellular ROS production and cytotoxicity of PPa/PPa-PEG2K NPs were investigated in mouse breast cancer cells(4T1).Compared with PPa solution,PPa/PCL-PEG2K NPs and PPa/PPa-PEG2K NPs showed stronger cell uptake.In addition.PPa/PPa-PEG2K NPs showed stronger cytotoxicity than PPa/PCL-PEG2K NPs because the modification of shell alleviated the ACQ effect to a certain extent,and improved intracellular ROS production capacity,thus exhibiting stronger cytotoxicity.The pharmacokinetic behavior and tissue distribution of PPa/PPa-PEG2K NPs were investigated in Sprague-Dawley(SD)rats and 4T1 tumor-bearing BALB/c mice,respectively.Compared with PPa solution,PPa/PCL-PEG2K NPs and PPa/PPa-PEG2K NPs significantly extended the circulation time in vivo and significantly increased the AUC of PPa.Compared with PPa/PCL-PEG2K NPs,PPa/PPa-PEG2K NPs exhibited better pharmacokinetic behavior and higher tumor accumulation due to the higher stability of PPa/PPa-PEG2K NPs.The antitumor activity and safety of PPa/PPa-PEG2K NPs were investigated in 4T1 tumor-bearing mice.Under laser irradiation,compared with PPa/PCL-PEG2K NPs and PPa solution,PPa/PPa-PEG2K NPs could exhibit better tumor accumulation and ROS generation ability so it is more effective to inhibit tumor proliferration.During the treatment,the body weight of all mice did not change significantly,the blood,liver and kidney function analysis showed no obvious abnormality between the groups,and the H&E tissue section staining showed no obvious damage to tissues and organs.In addition to carrier-free pure drug self-assembled NPs,carrier-free prodrug self-assembled NPs also showed unique advantages,especially homodimeric prodrug self-assembled NPs,which have ultra-high drug loading and greatly reduce the use of excipients.However,homodimeric prodrugs often exhibit poor assembly ability because of their strong symmetrical structure.In this paper,we first designed and synthesized an oxidation-sensitive carbataxel dimer prodrug(CTX-S-CTX),and found that it could not self-assemble into NPs in water.Interestingly,the co-assembled NPs of PPa and CTX-S-CTX could be spontaneously formed with the addition of PPa.In order to improve the stability of nanoparticles.20%DSPE-PEG2K was added to prepare the NPs(pCTX-S-CTX/PPa NPs)with uniform particle size and good stability.Moreover,PPa and CTX-S-CTX could co-assemble into NPs in a variety of proportions to optimize synergistic therapeutic efficiency.Therefore,this new self-assembly model of photosensitizer-driven dimeric prodrug not only solved the problem of poor self-assembly ability of homodimeric prodrug CTX-S-CTX,but also realized the flexible dose adjustment of photosensitizer and chemotherapeutic drug in the process of combined therapy.First,we investigated the co-assembly mechanism of the prodrug CTX-S-CTX and the photosensitizer PPa to form nanoparticles.Through molecular docking experiment,we found that there were multiple forces between CTX-S-CTX and PPa molecular.Then,after investigation of the intermolecular forces breaker and ultraviolet absorption and fluorescence spectra,we found in the process of assembly,hydrophobic effect and ?-?stacking effect occupies the leading role,and ?-cation and electricitic effect play the role of helper.The ACQ effect and oxidation sensitivity of pCTX-S-CTX/PPa NPs were investigated in release media containing various hydrogen peroxide(H2O2).The particle size of pCTX-S-CTX/PPa NPs increased with the increase of H2O2 concentration,suggesting NPs disintegrated gradually with the fracture of single sulfur bond in prodrug.In the process,the fluorescence of PPa was also recovered to a certain extent.Under laser excitation,with the increase of H2O2 concentration and incubation time,the singlet oxygen generation ability of PPa was gradually improved.These results demonstrated CTX-S-CTX had an excellent characteristic of oxidation-sensitive drug release,and the breakage of single sulfur bond in the dimeric prodrug structure could trigger the disintegration of the NPs,which significantly alleviated the ACQ effect of PPa.Then,the release of drug was investigated under different conditions(H2O2,laser irradiation and H2O2 and laser irradiation)with 30%anhydrous ethanol as the release medium.The results showed that the release of CTX increased gradually with the increase of H2O2 concentration or laser irradiation time.As expected,CTX release increased significantly when both H2O2 and laser irradiation conditions were present,compared to when H2O2 or laser irradiation were present alone.Therefore,PPa could not only drive CTX-S-CTX assembly to form NPs,but also synergically promote the oxidation-sensitive release of CTX under laser irradiation.The cell uptake and intracellular ROS production of pCTX-S-CTX/PPa NPs on 4T1 cells were investigated.pCTX-S-CTX/PPa NPs showed stronger cell uptake than PPa solution after 4 h of incubaition.pCTX-S-CTX/PPa NPs also showed desired ROS production with the extension of incubation time,due to the alleviation of ACQ effect of PPa.Subsequently,the cytotoxicity of pCTX-S-CTX/PPa NPs on 4T1 cells and human oral epidermoid cancer(KB)cells was investigated.Under laser irradiation,pCTX-S-CTX/PPa NPs exhibited significant synergistic cytotoxic effects of PDT and chemotherapy.In addition,the cytotoxicity of pCTX-S-CTX/PPa NPs on human normal liver cells(LO2)was also investigated.It is noteworthy that pCTX-S-CTX/PPa NPs had almost no toxicity on normal cells under dark condition,suggesting its good therapeutic selectivity.The pharmacokinetic behavior and tissue distribution of pCTX-S-CTX/PPa NPs were investigated in Sprague-Dawley(SD)rats and 4T1 tumor-bearing BALB/c mice,respectively.Compared with PPa solution,pCTX-S-CTX/PPa NPs significantly extended the circulation time in vivo,and had a higher AUC of PPa.In addition,pCTX-S-CTX/PPa NPs accumulated significantly more in tumor tissues than PPa solution.The antitumor activity and safety of pCTX-S-CTX/PPa NPs in vivo were investigated in 4T1 tumor-bearing BALB/c mice.The combination of PDT and chemotherapy displayed excellent synergistic therapeutic efficacy compared to the treatment mode alone.More importantly,pCTX-S-CTX/PPa NPs inhibited tumor growth more effectively under laser irradiation than PPa and CTX-S-CTX mixtures.During the period of treatment,there was no obvious change in body weight of mice treated with pCTX-S-CTX/PPa NPs.By contrast,the body weight of mice treated with CTX solution has a certain decline,suggesting NPs had lower systemic toxicity than solution agents.Moreover,the results of blood and kidney function analysis between groups had no obvious abnormalitie.There was no obvious damage in various tissues and organs no obvious damage about H&E staining.These results proved that pCTX-S-CTX/PPa NPs is a high-efficiency and low-toxicity nanomplatform.This simple and efficient co-assembly of photosensitizer and dimer prodrug solves the assembly problem of homodimer prodrug and provides a novel drug delivery model for cancer therapy. |
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