Cell-Penetrating Mitochondrion-Targeting Ligands for the Universal Delivery of Small Molecules,Proteins and Nanomaterials

Mitochondria are key organelles that perform vital cellular functions such as those related to cell survival and death. The targeted delivery of different types of cargos to mitochondria is a well-established strategy to study mitochondrial biology and diseases. Of the various existing mitochondrion-transporting vehicles, most suffer from poor cytosolic entry, low delivery efficiency, limited cargo types, and cumbersome preparation protocols, and none was known to be universally applicable for mitochondrial delivery of different types of cargos (small molecules, proteins, and nanomaterials). Herein, two new cell-penetrating, mitochondrion-targeting ligands (named Mito^Ligand) that are capable of effectively “tagging” small-molecule drugs, native proteins and nanomaterials are disclosed, as well as their corresponding chemoselective conjugation chemistry. Upon successful cellular delivery and rapid endosome escape, the released native cargos were found to be predominantly localized inside mitochondria. Finally, by successfully delivering doxorubicin, a well-known anticancer drug, to the mitochondria of HeLa cells, we showed that the released drug possessed potent cell cytotoxicity, disrupted the mitochondrial membrane potential and finally led to apoptosis. Our strategy thus paves the way for future mitochondrion-targeted therapy with a variety of biologically active agents.     

Mitochondrion targeting peptide-modified magnetic graphene oxide delivering mitoxantrone for impairment of tumor mitochondrial functions

In this study, we prepared mitochondrion targeting peptide-grafted magnetic graphene oxide (GO) nanocarriers for efficient impairment of the tumor mitochondria. The two-dimensional GOMNP-MitP nanosheets were synthesized by grafting magnetic γ-Fe₂O₃ to the surface of GO, followed by covalent modification of mitochondrion targeting peptide (MitP). GOMNP-MitP exhibited the high capacity of loading the anticancer drug mitoxantrone (MTX), and preferentially targeted the tumor mitochondria. With the aid of alternating magnetic field (AMF), the MTX-loading GOMNP-MitP released MTX to the mitochondria, severely impairing mitochondrial functions, including attenuation of ATP production, decrease in mitochondrial membrane potential (MMP), and further leading to activation of apoptosis. This study realized high-efficient mitochondrion-targeting drug delivery for anticancer therapy by two-dimensional nanoplatforms.     

Unique Use of Alkylation for Chemo‐Redox Activity by a PtIV Prodrug

Resistance towards chemotherapeutics displayed by cancer cells is a significant stumbling block against fruitful cisplatin‐based therapy. A unique dual‐acting chemotherapeutic modality, Platin‐B, a prodrug of cisplatin and pipobroman‐mimicking alkylating agent, was constructed to circumvent tumor resistance. Platin‐B exhibited a superior cytotoxicity profile in cisplatin‐resistant cancer cells. Enhanced activity and the ability to overcome cancer‐induced resistance of Platin‐B was related to adduct formation with intracellular glutathione, followed by the activity of Platin‐B on the mitochondria of cells, along with its conventional nuclear activity. Alkylating moieties present on Platin‐B enhanced its cellular and subcellular concentration and protected it from early drug sequestration by biological thiols.     

Mitochondria‐Targeted Cancer Therapy Using a Light‐Up Probe with Aggregation‐Induced‐Emission Characteristics

Subcellular organelle‐specific reagents for simultaneous tumor targeting, imaging, and treatment are of enormous interest in cancer therapy. Herein, we present a mitochondria‐targeting probe (AIE‐mito‐TPP) by conjugating a triphenylphosphine (TPP) with a fluorogen which can undergo aggregation‐induced emission (AIE). Owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, the AIE‐mito‐TPP probe can selectively accumulate in cancer‐cell mitochondria and light up its fluorescence. More importantly, the probe exhibits selective cytotoxicity for studied cancer cells over normal cells. The high potency of AIE‐mito‐TPP correlates with its strong ability to aggregate in mitochondria, which can efficiently decrease the mitochondria membrane potential and increase the level of intracellular reactive oxygen species (ROS) in cancer cells. The mitochondrial light‐up probe provides a unique strategy for potential image‐guided therapy of cancer cells.     

Photoactivatable prodrug for simultaneous release of mertansine and CO along with a BODIPY derivative as a luminescent marker in mitochondria: a proof of concept for NIR image-guided cancer therapy

Controlled and efficient activation is the crucial aspect of designing an effective prodrug. Herein we demonstrate a proof of concept for a light activatable prodrug with desired organelle specificity. Mertansine, a benzoansamacrolide, is an efficient microtubule-targeting compound that binds at or near the vinblastine-binding site in the mitochondrial region to induce mitotic arrest and cell death through apoptosis. Despite its efficacy even in the nanomolar level, this has failed in stage 2 of human clinical trials owing to the lack of drug specificity and the deleterious systemic toxicity. To get around this problem, a recent trend is to develop an antibody-conjugatable maytansinoid with improved tumor/organelle-specificity and lesser systematic toxicity. Endogenous CO is recognized as a regulator of cellular function and for its obligatory role in cell apoptosis. CO blocks the proliferation of cancer cells and effector T cells, and the primary target is reported to be the mitochondria. We report herein a new mitochondria-specific prodrug conjugate (Pro-DC) that undergoes a photocleavage reaction on irradiation with a 400 nm source (1.0 mW cm⁻²) to induce a simultaneous release of the therapeutic components mertansine and CO along with a BODIPY derivative (BODIPY(PPH₃)₂) as a luminescent marker in the mitochondrial matrix. The efficacy of the process is demonstrated using MCF-7 cells and could effectively be visualized by probing the intracellular luminescence of BODIPY(PPH₃)₂. This provides a proof-of-concept for designing a prodrug for image-guided combination therapy for mainstream treatment of cancer.

Simultaneous release of two therapeutic reagents, mertansine and CO through photo-induced cleavage of a mitochondria-specific prodrug with improved drug efficacy.     

Synthesis and preliminary cytotoxicity study of a cephalosporin-CC-1065 analogue prodrug

Background  

Antibody-directed enzyme prodrug therapy (ADEPT) is a promising new approach to deliver anticancer drugs selectively to tumor cells. In this approach, an enzyme is conjugated to a tumor-specific antibody. The antibody selectively localizes the enzyme to the tumor cell surface. Subsequent administration of a prodrug substrate of the enzyme leads to the enzyme-catalyzed release of the free drug at the tumor site. The free drug will destroy the tumor cells selectively, thus, reducing side effects.     

GSH/ROS Dual-Responsive Supramolecular Nanoparticles Based on Pillar[6]arene and Betulinic Acid Prodrug for Chemo–Chemodynamic Combination Therapy

Chemodynamic therapy (CDT) based on intracellular Fenton reactions is attracting increasing interest in cancer treatment. A simple and novel method to regulate the tumor microenvironment for improved CDT with satisfactory effectiveness is urgently needed. Therefore, glutathione (GSH)/ROS (reactive oxygen species) dual-responsive supramolecular nanoparticles (GOx@BNPs) for chemo–chemodynamic combination therapy were constructed via host–guest complexation between water-soluble pillar[6]arene and the ferrocene-modified natural anticancer product betulinic acid (BA) prodrug, followed by encapsulation of glucose oxidase (GOx) in the nanoparticles. The novel supramolecular nanoparticles could be activated by the overexpressed GSH and ROS in the tumor microenvironment (TME), not only accelerating the dissociation of nanoparticles—and, thus, improving the BA recovery and release capability in tumors—but also showing the high-efficiency conversion of glucose into hydroxyl radicals (·OH) in succession through intracellular Fenton reactions. Investigation of antitumor activity and mechanisms revealed that the dramatic suppression of cancer cell growth induced by GOx@BNPs was derived from the elevation of ROS, decrease in ATP and mitochondrial transmembrane potential (MTP) and, finally, cell apoptosis. This work presents a novel method for the regulation of the tumor microenvironment for improved CDT, and the preparation of novel GSH/ROS dual-responsive supramolecular nanoparticles, which could exert significant cytotoxicity against cancer cells through the synergistic interaction of chemodynamic therapy, starvation therapy, and chemotherapy (CDT/ST/CT).     

Mitochondria targeted cancer therapy using ethidium derivatives

In this study, we reported on two novel fluorescent ethidium derivatives of MTP1 and MTP2 for selective and efficient cancer therapy. MTP1 and MTP2 exhibited cancer cell targeting and subsequent mitochondria targeting and imaging abilities. Moreover, both MTP1 and MTP2 would induce mitochondria depolarization and so along with a series of cascaded biochemical effects including the reduction of ATP production, destruction of intracellular redox potential balance and release of mitochondria cytochrome C (Cyt C), which could finally trigger caspase-dependent cell apoptosis. More interestingly, both MTP1 and MTP2 demonstrated significant cancer suppression abilities in vitro and in vivo, which presented a new paradigm for the development of unique anti-cancer agent candidates in precise and efficient cancer theranostics.     

Redirecting Cisplatin and Doxorubicin to Mitochondria Affords Highly Effective PlatinumIV Prodrug Against Triple Negative Breast Cancer

A mitochondria targeting dual-action platinum^IV prodrug exhibits high anticancer activity in triple negative breast cancer cells. The complex intervenes in several cellular processes including DNA damage, perturbation of mitochondrial bioenergetics and induction of necrosis to kill cancer cells.     

Fluorescent Coumarin–Artemisinin Conjugates as Mitochondria‐Targeting Theranostic Probes for Enhanced Anticancer Activities

Mitochondria‐targeting theranostic probes that enable the simultaneously reporting of and triggering of mitochondrial dysfunctions in cancer cells are highly attractive for cancer diagnosis and therapy. Three fluorescent mitochondria‐targeting theranostic probes have been developed by linking a mitochondrial dye, coumarin‐3‐carboximide, with a widely used traditional Chinese medicine, artemisinin, to kill cancer cells. Fluorescence images showed that the designed coumarin–artemisinin conjugates localized mainly in mitochondria, leading to enhanced anticancer activities over artemisinin. High cytotoxicity against cancer cells correlated with the strong ability to accumulate in mitochondria, which could efficiently increase the intracellular reactive oxygen species level and induce cell apoptosis. This study highlights the potential of using mitochondria‐targeting fluorophores to selectively trigger and directly visualize subcellular drug delivery in living cells.     

5‐Methoxytryptophan‐dependent inhibition of oral squamous cell carcinoma metastasis

The metastatic status of oral cancer is highly associated with the overall survival rate of patients. Previous studies have revealed that the endogenous tryptophan metabolite 5‐methoxytryptophan (5‐MTP) can downregulate cyclooxygenase‐2 expression; suppress tumor proliferation, migration, and invasion; and reduce the tumor size. To improve the understanding of the molecular mechanisms involved in the regulation of 5‐MTP in the tumorigenesis of oral cancer, we conducted a comparative wound healing and transwell invasion assays. Our results revealed that 5‐MTP reduce oral cancer cell migration and invasion ability. In addition, the results of an in vivo assay demonstrated that the growth of primary tumors was significantly inhibited by 5‐MTP in OC3 oral cancer cells and in invasive OC3‐I5 oral cancer cells. Moreover, enlarged spleens were observed in OC3‐I5‐implanted severe combined immunodeficiency mice although 5‐MTP can inhibit spleen enlargement. Through comparative proteomics, we identified 32 differentially regulated protein spots by using 2D‐DIGE/MALDI‐TOF MS analyses. Some of the differentially regulated proteins such as amadillo‐repeat‐containing X‐linked protein 1, phosphoglycerate kinase 1, tropomyosin alpha‐1, and tropomyosin alpha‐4 may be associated with the 5‐MTP‐dependent inhibition of oral cancer growth and metastasis. We conclude that 5‐MTP plays a crucial role in inhibiting in vitro and in vivo cancer invasion and metastasis.     

Specific redistribution of cell-penetrating peptides from endosomes to the cytoplasm and nucleus upon laser illumination

Cell-penetrating peptides (CPPs), once postulated to cross cell membranes in a non-endocytic, non-energy-dependent process, have since been found to accumulate in vesicles in live mammalian cells. In this study, we show that it is possible to use laser light from a confocal microscope to cause labeled peptide-conjugated CPPs to redistribute from vesicles into the cytoplasm and nucleus of cells. Following redistribution, the cells are found to be biologically responsive, and they retain morphology for several hours. It was possible to initiate redistribution of both fluorescein- and Alexa633-labeled peptides by selective irradiation of one of the fluorophores. These peptides could potentially be used as tracers to selectively deliver cargo biomolecules into cells by laser illumination using a standard fluorescence confocal microscope.     

A highly X-ray sensitive iridium prodrug for visualized tumor radiochemotherapy

Concomitant treatment of radiotherapy and chemotherapy is widely used in cancer therapy. The search for highly efficient radiochemotherapy drugs for tumor targeting therapy under image-guiding is of considerable interest. Herein we report an Ir-based prodrug Ir-NB with high sensitization efficiency for in vivo tumor microenvironment responsive cancer-targeted bioimaging radiochemotherapy. To the best of our knowledge, the sensitivity enhancement ratio (SER) of the Ir-NB prodrug is the highest among those reported for radiotherapy metal complex drugs. From detailed action mechanism study, we provide evidence that the prodrug is effectively suppresses the tumor growth through inducing mitochondrial dysfunction, and eventually amplifies the apoptotic signal pathway. This study provides an approach for the development of cancer theranostic agents for tumor radiotherapy.

A highly X-ray sensitive molecular prodrug, Ir-NB, was reported for visualized tumor radiochemotherapy. To our knowledge, the sensitivity enhancement ratio of the prodrug is the highest among the reported radiotherapy metal complexes drugs.     

Dual Diomarkers Triggered Prodrugs for Precise Treatment of Melanoma:Design,Synthesis and Activities

Targeted prodrug strategy, which utilizes the endogenous biomarkers in cancer cells as activators to release the active drug, has been well established either in the fundamental research or the clinical treatment. However, many prodrugs suffer from safety concern due to "off-target activation". Dual or multiple biomarkers triggered prodrug may provide an effective strategy to overcoming the "off-target effect". Melanoma cells have both high levels of reactive oxygen species(ROS) and tyrosinase(TYR), which makes them significantly different from other tumor cells and normal cells. Here we reported a series of quinazolinone-aryl boronic acid/ester-based prodrugs, which can be activated by the cascade of ROS and TYR and selectively kill melanoma cells. The structure-activity relationship(SAR) analysis revealed that mitochondria-targeting property was vital for their cytotoxicity and the dual activated effector played a significant role in their selectivity towards melanoma cells. Among these candidates, compound 4b showed the highest toxicity to B16, leading to an imbalance of the redox system in melanoma cells, causing mitochondrial DNA damage, and then promoting melanoma cells death.     

Simultaneous Application of Photothermal Therapy and an Anti‐inflammatory Prodrug using Pyrene–Aspirin‐Loaded Gold Nanorod Graphitic Nanocapsules

Photothermal therapy (PTT) has been extensively developed as an effective approach against cancer. However, PTT can trigger inflammatory responses, in turn simulating tumor regeneration and hindering subsequent therapy. A therapeutic strategy was developed to deliver enhanced PTT and simultaneously inhibit PTT‐induced inflammatory response. 1‐Pyrene methanol was utilize to synthesize the anti‐inflammatory prodrug pyrene–aspirin (P‐aspirin) with a cleavable ester bond and also facilitate loading the prodrug on gold nanorod (AuNR)‐encapsulated graphitic nanocapsule (AuNR@G), a photothermal agent, through π–π interactions. Such AuNR@G‐P‐aspirin complexes were used for near‐infrared laser‐triggered photothermal ablation of solid tumor and simultaneous inhibition of PTT‐induced inflammation through the release of aspirin in tumor milieu. This strategy showed excellent effects in vitro and in vivo.     

Mitochondrion-anchoring AIEgen with Large Stokes Shift for Imaging-guided Photodynamic Therapy

Photosensitizers that can target and accumulate in mitochondria are expected to achieve good therapeutic effects in photodynamic therapy,as mitochondria are the energy generation factory in cells.Herein,we designed and synthesized a novel mitochondrion-targeting photosensitizer TPC-Py with aggregation-induced emission characteristics for image-guided photodynamic therapy.TPC-Py possessed an efficient production of 1O2,with a quantum yield of 11.65%,upon mild white light irradiation(6 mW/cm²).TPC-Py exhibited good biocompatibility under dark condition,but showed remarkable cytotoxicity towards human cervical carcinoma(HeLa)cells with a half maximal inhibitory concentration(IC50)of 3.2μmol/L when exposed to white light irradiation(14.4 J/cm²).In addition,the Stokes shift of TPC-Py was as high as 150 nm,so that it could prevent self-absorption and increase the signal-to-noise ratio of fluorescence imaging.The excellent performance of TPC-Py makes it a promising candidate in imaging-guided clinical PDT for cancer in the near future.     

pIL-12 delivered by polymer based nanovector for anti-tumor genetherapy

Finding more effective and safe non-viral vectors to transfer genes into cancer cells has become the key of immune gene therapy for cancer. Herein a triblock compound MPEG₂₀₀₀–PDLLA₄₀₀₀–MPEG₂₀₀₀ modified by cationic liposome DOTAP was used as a non-viral vector DOTAP/MPEG₂₀₀₀–PDLLA₄₀₀₀–MPEG₂₀₀₀ (DMPM) to effectively transfer interleukin (IL)-12 plasmid (pIL-12) into tumor tissue. IL-12 produced by transfected tumor cells successfully inducing lymphocyte proliferation and promoting interferon-γ (IFN-γ) secretion, which resulted in tumor cells death. The ability of DMPM to transfer pIL-12 and the immune effect induced by IL-12 in cells had been explored. The anti-tumor effect, mechanism and safety of pIL-12/DMPM in mice cancer model were investigated in this study. Our results showed that the pIL-12 transferred by DMPM was highly expressed both in CT26 cells and B16-F10 cells. IL-12 expressed in the culture supernatant of transfected tumor cells stimulated lymphocyte proliferation and promoted IFN-γ secretion. The experimental result confirmed that pIL-12/DMPM therapy significantly reduced tumor growth in mice model. We designed the nanocomposite DMPM to deliver pIL-12 for cancer treatment and explored its therapeutic efficacy and the underlying anti-tumor mechanism. Our study suggested pIL-12 loaded by DMPM complex would be an effective strategy for cancer treatment.     

Multi‐Stimuli‐Responsive Polymeric Prodrug for Enhanced Cancer Treatment

Accomplishing efficient delivery of a nanomedicine to the tumor site will encounter two contradictions as follows: 1) a contradiction between prolonged circulation time and endocytosis by cancer cells; 2) a dilemma between the stability of nanomedicine during blood circulation and intracellular drug release. While developing a nanomedicine which can solve the above two contradictions simultaneously is still a challenge, here, a multi‐stimuli‐responsive polymeric prodrug (PLys‐co‐(PLys‐DA)‐co‐(PLys‐SS‐PTX))‐b‐PLGLAG‐mPEG (P‐PEP‐SS‐PTX‐DA) is synthesized which is multi‐sensitive to overexpressed matrix metalloproteinase‐2 (MMP‐2), low pH, and high concentration of glutathione in tumors. The P‐PEP‐SS‐PTX‐DA can be dePEGylated and reversed from negative at normal physiological pH to positive charge at tumor extracellular microenvironment; in this way, it can solve the contradiction between prolonged circulation time and endocytosis by cancer cells. Owing to the high reductive conditions in cancer cells, P‐PEP‐SS‐PTX‐DA is ruptured to release paclitaxel (PTX) intracellular efficiently; therefore, it can resolve the dilemma between the stability of nanomedicine during blood circulation and intracellular drug release. These indicate that the multi‐stimuli‐responsive polymeric prodrug has potential application prospects in drug delivery and cancer therapy.     

Preparation of a Camptothecin Prodrug with Glutathione‐Responsive Disulfide Linker for Anticancer Drug Delivery

We present here a novel camptothecin (CPT) prodrug based on polyethylene glycol monomethyl ether‐block‐poly(2‐methacryl ester hydroxyethyl disulfide‐graft‐CPT) (MPEG‐SS‐PCPT). It formed biocompatible nanoparticles (NPs) with diameters of approximately 122 nm with a CPT loading content as high as approximately 25 wt % in aqueous solution. In in vitro release studies, these MPEG‐SS‐PCPT NPs could undergo triggered disassembly and much faster release of CPT under glutathione (GSH) stimulus than in the absence of GSH. The CPT prodrug had high antitumor activity, and another anticancer drug, doxorubicin hydrochloride (DOX ? HCl), could also be introduced into the prodrug with a high loading amount. The DOX ? HCl‐loaded CPT prodrug could deliver two anticancer drugs at the same time to produce a collaborative cytotoxicity toward cancer cells, which suggested that this GSH‐responsive NP system might become a promising carrier to improve drug‐delivery efficacy.     

The energy blocker inside the power house: mitochondria targeted delivery of 3-bromopyruvate

A key hallmark of many aggressive cancers is accelerated glucose metabolism. The enzymes that catalyze the first step of glucose metabolism are hexokinases. Elevated levels of hexokinase 2 (HK2) are found in cancer cells, but only in a limited number of normal tissues. Metabolic reprogramming of cancer cells using the energy blocker 3-bromopyruvate (3-BP), which inhibits HK2, has the potential to provide tumor-specific anticancer agents. However, the unique structural and functional characteristics of mitochondria prohibit selective subcellular targeting of 3-BP to modulate the function of this organelle for therapeutic gain. A mitochondria-targeted gold nanoparticle (T-3-BP-AuNP), decorated with 3-BP and delocalized lipophilic triphenylphosphonium cations to target the mitochondrial membrane potential (Δψ _m), was developed for delivery of 3-BP to cancer cell mitochondria by taking advantage of the higher Δψ _m in cancer cells compared to normal cells. In vitro studies demonstrated an enhanced anticancer activity of T-3-BP-AuNPs compared to the non-targeted construct NT-3-BP-AuNP or free 3-BP. The anticancer activity of T-3-BP-AuNPs was further enhanced upon laser irradiation by exciting the surface plasmon resonance band of AuNP and thereby utilizing a combination of 3-BP chemotherapeutic and AuNP photothermal effects. The lower toxicity of T-3-BP-NPs in normal mesenchymal stem cells indicated that these NPs preferentially kill cancer cells. T-3-BP-AuNPs showed an enhanced ability to modulate cancer cell metabolism by inhibiting glycolysis as well as demolishing mitochondrial oxidative phosphorylation. Our findings demonstrate that concerted chemo-photothermal treatment of glycolytic cancer cells with a single NP capable of targeting mitochondria, mediating simultaneous release of a glycolytic inhibitor and photothermal ablation, may have promise as a new anticancer therapy.