Development Of Photoactivatable Small-molecule Prodrug Nanoassemblies For Chemo/Photodynamic Therapy Against Melanoma Tumors

Chemotherapy is one of the most effective approaches in clinical tumor treatment.Cabazitaxel(CTX),as a semisynthetic taxane,is a new antineoplastic drug with low P-glycoprotein(P-gp)affinity,thereby overcoming drug resistance induced by paclitaxel(PTX).However,the low solubility and remarkable systemic toxicity limit its clinical application.In recent years,the development of nanomedicine has made it possible to improve the physicochemical properties of medicines and to establish safe and efficient drug delivery systems(DDSs).Currently,polymer-based nano DDS,which is widely studied,often requires complex synthesis scheme so that the quality is poor controllability.In addition,a large quantity of polymer matrices introduced into the nanosystem makes it difficult to increase the drug loading capacity.Therefore,how to design a novel nanocarrier for safe,efficient,economical and controllable drug delivery,and ultimately achieving clinical translation,is an urgent scientific problem.From this,simple and green"Carrier-free nanoassemblies"nanodrugs without additional polymer materials are gradually favored by researchers.Based on supramolecular self-assembly,carrier-free nanotechnology has been proved by more and more studies to have high drug loading capacity,lower systemic toxicity,accurately characterized chemical structure,and unambiguously visualized nano-assembly process via molecular dynamics simulation.Among them,the carrier-free nanodrugs established based on drug dimer or PUFAylation technology can achieve high-efficiency drug loading over polymer or liposome-based encapsulation strategies,as well as enhance the stability and targeting ability over free drug combination.Since chemotherapy alone has the difficulty to achieve good results in the treatment of malignant tumors,the current trend has gradually paid attention to combination therapy.Among them,with the advantages of minimally invasive property,reduced side effects and spatiotemporal controllability,photodynamic therapy(PDT)is rationally paired with chemotherapy to enhance antitumor efficacy.Exploiting reactive oxygen species(ROS)generated by photodynamic reaction to design ROS-responsive smart prodrugs and further introducing them into the preparation of nanosystems,can increase the drug stability in the circulation time and the sensitivity in the site of disease,which results in remarkable superadditive(namely"1+1>2")effects of smart DDS,greater than any single therapy or their theoretical combination.Based on the background,focusing on the bottleneck problems of cabazitaxel,such as poor water solubility,remarkable systemic toxicity,and low drug loading capacity in the polymeric system,the research introduced the"Carrier-free self-assembly"strategy for nanofabrication.In addition,the research further adopted the smart small-molecule prodrugs design and combination therapy for the development of photoactivatable carrier-free nanodrugs based on the cabazitaxel dimer or PUFAylation technology.The precise and controllable nanodesign were organically combined with multifunctional drug delivery strategies to explore the curative effects of novel smart DDSs using malignant melanoma as a model.The first part reported a photosensitizer-stabilized,supramolecular photoactivatable nanoassembly based on cabazitaxel dimer.Two anticancer cabazitaxel(CTX)drugs rational bridged via a thioketal linkage to generate a TKdC prodrug,which subsequently coassembled with a photosensitizer,chlorin e6(Ce6).The nanoassembly(termed psTKdC NAs)was stabilized by multiple intermolecular interactions,including hydrogen bonding,π-πstacking,and hydrophobic interactions.Firstly,the drug ratio of nanoassembly was optimized by the particle size and the drug encapsulation efficiency.After that,through the morphological observation,UV-vis/NIR absorption spectra and HPLC analysis,psTKdC NAs was demonstrated that not only had high drug loading capacity(>90%),but also could effectively respond to laser activation and cleave the thioketal(TK)linkage for on-demand CTX release,ultimately promoting the reductive transformation of nanosize.In vitro experiments had shown that psTKdC NAs could increase intracellular uptake and ROS level,further inducing DNA oxidative damage and triggering CTX release for tubulin depolymerization inhibition,and eventually led to tumor cell apoptosis or necrosis.Moreover,in vivo pharmacokinetics indicated that psTKdC NAs improved the pharmacokinetic properties ofCTX as well as increased the accumulation of drugs in tumor tissues to a certain extent,compared to the free CTX in Jevtana-mimicking formulation in clinic.Then,patient-derived xenograft(PDX)model of malignant melanoma was established to evaluate the synergistic therapy of psTKdC NAs in vivo.During the therapeutic period,the tumor volume,body weight and survival rate were recorded.And the results demonstrated that administration of psTKdC NAs followed by laser irradiation produced durable tumor regression,with the tumors being completely eradicated in 50%PDX model.In addition,the in vivo safety evaluation found that psTKdC NAs with good blood compatibility,could significantly reduce the myelosuppression as well as liver and kidney toxicity induced by the free drug combination.Building on the PUFAylation technology of active agents,the second part exploited naturally abundant and commercially available polyunsaturated fatty acids(PUFAs)to finely tuned the flexibility and the amphiphilicity of drug molecules,and thereby designed a photoactivatable small-molecule prodrug nanoassembly named PSPC NAs.The PSPC NAs were co-assembly of the LTK-CTX prodrug,in whichα-linolenic acid(LNA)was ligated to CTX via a self-immolative thioketal linker,and LNA-conjugated photosensitizer Ce6(termed L-Ce6).Furthermore,both PUFAylated prodrugs were capable of either forming colloidal-stable nanoassemblies individually or coassembling into cocktail nanoassemblies over a wide range of molar ratios of the prodrugs without any excipients.Morphological observation and HPLC analysis found that,compared with the nondegradable group(nPSPC NAs),the LTK-CTX prodrug in PSPC NAs was effectively degraded upon laser activation,thus promoting the reductive transformation of nanosize.In vitro experiments had indicated that ROS generated by L-Ce6 not only efficiently induced DNA damage,markedly decreased mitochondrial membrane potential,but also spontaneously degraded thioketal bond to release CTX via bystander effect,thereby exerting a synergistic effect of PDT and chemotherapy.Then,in vivo distribution revealed that extended in vivo circulation time and favorable tumor-targeting were achieved using PUFAylation technology through the simple bioconjugation of therapeutic drugs with a natural lipophilic fatty acid.In the A375 human melanoma xenograft model,when given the same dose of different formulations(5 mg/kg cabazitaxel-equivalent dose),PSPC NAs and fCTX/fCe6 groups produced similarly potent antitumor activity.Unfortunately,fCTX/fCe6 plus laser treatment exhibited significant toxicity,leading to a substantial drop in the body weight of mice(～24%).After that,the efficacy of PSPC NAs and fCTX/fCe6 at relatively safe doses were further validated.Disappointingly,reduced doses of the fCTX/fCe6 lost the potency.Interestingly,treatment with PSPC NAs(15mg/kg cabazitaxel-equivalent dose)resulted in tumor shrinkage,and the average volume of the tumors were 23.4±15.8 mm~3 at the end of the experiment,which were substantially smaller than the tumors treated with the fCTX/fCe6(V=958.3±389.9 mm~3,2 mg/kg cabazitaxel-equivalent dose).In addition,in the PDX model,PSPC NAs also showed better antitumor efficacy than the free drug combination.The body weight,survival rate,the numbers of white blood cells,and serum biochemical analysis of liver and kidney in ICR mice were used to evaluate the in vivo safety of PSPC NAs at high doses.The results displayed that PSPC NAs dramatically improved drug tolerability in animals,expanding its therapeutic window in clinical practice.The research introduced the"Carrier-free self-assembly"strategy,as well as adopted smart prodrug design and combination therapy for the development of photoactivatable carrier-free nano DDS based on the cabazitaxel dimer and PUFAylation technology.The nanoassemblies improved the drug physicochemical properties,realized high drug loading capacity and the controlled release under laser activation,thus exhibiting a synergistic antitumor effect and good in vivo safety in a malignant melanoma model.Based on a simple and green carrier-free drug delivery technology,the research organically combined spatiotemporally controllable nanodesign with multifunctional drug delivery strategies,providing new ideas for the clinical translation of novel smart DDSs and the efficient treatment of malignant tumors.