Study On Preparation Of 5-aminolevulinic Acid Nanoprodrug And Its Application In Sonodynamic Treatment Of Skin Cancer

Background:Skin cancer is a common form of cancer with an increasing incidence worldwide.Cutaneous melanoma and cutaneous squamous cell carcinoma are the two kinds of skin malignant tumors with the highest degree of malignancy and mortality.In addition to the traditional surgical treatment,photodynamic therapy is developing rapidly in the clinical treatment of skin tumors,and has gained more and more applications and affirmation.However,photodynamic therapy cannot be used to treat large tumors due to insufficient penetration depth.Therefore,it is urgent to break through the treatment bottleneck of skin tumor and break the limitation of photodynamic therapy.Sonodynamic therapy（SDT）is similar to the principle of PDT local produce reactive oxygen species are used to treat with the characteristics of a noninvasive and precision and ultrasonic have deeper tissue penetration ability,that makes the properties of acoustic power treatment compared with photodynamic therapy in the field of tumor treatment is more practical,is expected to be used for the treatment of skin cancer.However,the therapeutic effect of sonodynamics is affected by many factors.For example,（1）High concentrations of reduced glutathione（GSH）in tumor cells form a reductive tumor microenvironment that reduces reactive oxygen species（ROS）produced during sonodynamic therapy;（2）Lack of sound sensitive agents to meet biological safety.Although acoustic dynamic therapy has been reported by delivering organic molecules such as protoporphyrin（Pp IX）or hematoporphyrin,these acoustic agents often have concomitant phototoxicity and bring safety risks to patients.Therefore,it is very important to design the sonodynamic therapy to change the tumor microenvironment and satisfy the biosafety.Strategies to consume GSH through chemical reactions to improve the reductive tumor microenvironment have been widely reported.Recent studies have shown that SO2,which is often used in gas therapy,can react with GSH as an oxidizing gas molecule to weaken the reductive tumor microenvironment and increase the level of intracellular reactive oxygen species（ROS）,which not only improves the reductive tumor microenvironment but also achieves the purpose of therapy.5-aminolevulinic acid（ALA）is an FDA-approved drug that specifically biosyntheses protoporphyrin IX（Pp IX）in tumor cells for the treatment of PDT.ALA has been used as a safe photosensitizer in the treatment of a variety of skin related diseases.When ALA was used for local administration,PDT was only used for superficial skin tumors due to its poor permeability and limited light penetration.When ALA is used for systematic administration,it is difficult to achieve the desired therapeutic effect due to the fast clearance of small molecules in the body and the difficulty to accumulate at the tumor site.Fortunately,Pp IX has been widely reported as a sound sensitizer for SDT,and we anticipate that the inclusion of ALA in nanodrugs for SDT will solve the current application dilemma,while the safety of ALA provides a biosafety sound sensitizer for SDT of skin tumors.In view of this,in this paper,we combine the respective advantages of SO2 and ALA to prepare two-in-one nanoprodrugs.The prodrug utilizes the enhanced permeability and retention effect（EPR）of nanomedicine to enhance tumor accumulation.The released ALA could be transferred into protoporphyrin IX（Pp IX）in tumor cells for SDT and photoacoustic imaging.Meanwhile,the release of SO2and depletion of GSH resulted from the reaction between DOA and GSH could sharply increase the intracellular ROS level in cancer cells.Furthermore,the P-DOA NPs achieved excellent tumor inhibition under the guidance of real-time photoacoustic imaging in the in vivo melanoma and SCC models.c It is expected that the two-in-one nano prodrug can stimulate systemic immune response,which will inhibit the metastasis of melanoma,hopefully solve the problem of high recurrence and high metastasis rate of melanoma,and provide new ideas for clinical treatment of melanoma.The specific research content is as follows.Objective:By integrating the antitumor advantages of SO2 and ALA,a GSH responsive two-in-one prodrug molecule was synthesized,and nano prodrug was prepared by assembly.To explore the enhanced SDT effect of the two-in-one nano prodrug in vivo and in vitro,to verify its tumor suppressive effect and explore the related mechanism in melanoma and squamous cell carcinoma skin tumor models,and to further the influence of the nano prodrug on systemic immunity,aiming to provide a new strategy for the treatment of skin malignant tumors.Methods:1.DOA was synthesized by coupling reaction between DNs and ALA,and then co-assembled with methoxy poly（ethylene glycol）-b-poly（L-lysine）（m PEG-b-PLL）to generate p-DOA NPs.The successful synthesis of DOA was verified by ¹H NMR,¹³C NMR and mass spectrometry.The size and morphology of P-DOA NPs were verified by dynamic light scattering（DLS）,transmission electron microscopy（TEM）and scanning electron microscopy（SEM）.The drug loading and encapsulation rate of DOA were measured by ICP-MS.Finally,the reduction responsiveness of DOA and P-DOA NPs was detected by ¹H NMR and DLS,respectively.The release capacity of SO2 was determined qualitatively and quantitatively by SO2test paper and fluorescence probe.Finally,GSH consumption capacity was detected with the kit.2.In vitro experiments:The conversion of ALA into Pp IX after responsive release of nanomedicine was qualitatively and quantitatively detected by confocal laser,flow cytometry and enzyme-plate analyzer respectively.The expression of SO2was detected by DEACA probe.Intracellular GSH consumption was detected by GSH kit.Intracellular ROS generation was detected by DCFH-DA probe.Finally,MTT assay was used to verify the anti-tumor ability of the nanomedicine against B16F10 and SCC cells in vitro.3.In vivo experiment:In vivo distribution and biosafety of nanoparticles were evaluated by constructing a mouse melanoma model.Photoacoustic imaging was used to observe the accumulation of drugs in melanoma and SCC tumor sites and guide the treatment,and further explore the tumor suppressive effect of drugs on the two skin tumor models.Finally,H&E,TUNEL and ROS staining of tumor tissues was used to explore the mechanism of anti-tumor action.4.Immune-related experiments:Flow cytometry was performed on immune cells in tumor tissues and lymph nodes after tumor suppressive treatment in melanoma model to verify the activation of systemic immunity and the regulation of immune microenvironment by nano drugs;Through immunofluorescence staining of tumor tissue,the anti-tumor immune mechanism of nano drugs was explored.Results:1.Dual prodrug molecule DOA was successfully synthesized and nano prodrug P-DOA NPs was successfully prepared.TEM and SEM showed that p-DOA NPs were uniformly distributed and spherical.The particle size of P-DOA NPs was109nm by dynamic light scattering method.The drug loading and encapsulation rate of P-DOA NPs were 27%and 55%respectively.P-DOA NPs will lose its spherical structure and disintegrate and release SO2 in the presence of mercaptan,while DOA releases ALA in the presence of mercaptan.The GSH kit verified that P-DOA NPs consumed 20%of GSH in vitro.2.In vitro experiments have proved that P-DOA NPs can enter tumor cells,and under the action of intracellular GSH,not only ALA can be responsively released and converted into Pp IX,but also SO2 can be released.The GSH kit confirmed that P-DOA NPs can consume 60%of GSH in tumor cells after 6 hours,and significantly increase intracellular ROS production level.MTT assay showed that P-DOA NPs had good antitumor effect on both skin tumor cells.3.In vivo experiments have proved that P-DOA NPs has good biosafety and can accumulate significantly in tumor sites.Under the guidance of photoacoustic imaging,it has good tumor inhibition effect on mouse melanoma model and SCC model.4.Immune-related experiments have proved that P-DOA NPs can activate systemic immunity and regulate immune microenvironment under the action of ultrasound,and can induce immunogenic death（ICD）of melanoma cells.Conclusion:In this experiment,double prodrug molecule DOA was successfully synthesized and nano prodrug P-DOA NPs was successfully assembled.The two-in-one nanoprodrug P-DOA NPs responds to the release of ALA by intracellular glutathione and the production of SO2.The release of ALA can specifically produce Pp IX at the tumor site,which can be used to guide treatment by photoacoustic imaging or as a sonodynamic therapy agent.The resulting SO2 can reduce tumor cell reducing TME by consuming GSH,increase ROS level and enhance SDT effect.In vivo experiments showed that P-DOA NPs had good antitumor effect on melanoma and SCC models under US irradiation.Immune-related experiments have proved that P-DOA NPs can promote the maturation of DC cells,present tumor antigens to T cells,activate the immune system,change the immunosuppressive tumor microenvironment,and achieve long-term tumor inhibition.In conclusion,P-DOA NPs,as a common prodrug of ALA and SO2,provides a new idea for clinical SDT study of tumors.