Platin-C containing nanoparticles: a recipe for the delivery of curcumin-cisplatin combination chemotherapeutics to mitochondria.

The success story of cisplatin spans over six decades now and yet it continues to be the key player in most chemotherapeutic regimens. Numerous efforts have been made to improve its efficacy, address its shortcomings, and overcome drug resistance. One such strategy is to develop new platinum(IV)-based prodrugs with functionally active ligands to deliver combination therapeutics. This strategy not only enables the drug candidate to access multiple drug targets but also enhances the kinetic inertness of platinum complexes and thereby ensures greater accumulation of active drugs at the target site. We report the synthesis of Platin-C, a platinum(IV)-based cisplatin prodrug tethered to the active component of ancient herbal medicine, curcumin, as one of the axial ligands. This combination complex showed improved chemotherapeutic efficacy in cisplatin resistant A2780/CP70 cell lines compared with the individual components. An amine-terminated biodegradable polymer was suitably functionalized with the triphenylphosphonium (TPP) cation to obtain a mitochondria-directed drug delivery platform. Quantification of Platin-C loading into these NPs using complementary techniques employing curcumin optical properties in high-performance liquid chromatography and platinum-based inductively coupled plasma mass spectrometry evidenced efficacious payload incorporation resulting in functional activities of both the components. Stability studies for a period of one week indicated that the NPs remain stable, enabling substantial loading and controlled release of the prodrug. The targeting nanoparticle (NP) platform was utilized to deliver Platin-C primarily in the mitochondrial network of cancer cells as monitored using confocal microscopy employing the green fluorescence of the curcumin pendant. Our studies showed that amine terminated NPs were relatively less efficient in their ability to target mitochondria despite being positively charged. This re-validated the importance of lipophilic positively charged TPP surface functionalities to successfully target cellular mitochondria. We validated the capabilities of Platin-C and its mitochondria-targeting nanoparticles towards inflicting mitochondria-directed activity in cisplatin-sensitive and cisplatin-resistant cell lines. Furthermore, our studies also demonstrated the effectiveness of Platin-C incorporated targeting NPs in attenuating cellular inflammatory markers by utilizing the curcumin component. This study advances our understanding of the cisplatin prodrug approach to combine chemotherapeutic and inflammatory effects in accessing combinatory pathways.

Dual-Targeted Synthetic Nanoparticles for Cardiovascular Diseases.

Atherosclerosis is one of the world's most aggressive diseases, claiming over 17.5 million lives per year. This disease is usually caused by high amounts of lipoproteins circulating in the blood stream, which leads to plaque formation. Ultimately, these plaques can undergo thrombosis and lead to major heart damage. A major contributor to these vulnerable plaques is macrophage apoptosis. Development of nanovehicles that carry contrast and therapeutic agents to the mitochondria within these macrophages is attractive for the diagnosis and treatment of atherosclerosis. Here, we report the design and synthesis of a dual-targeted synthetic nanoparticle (NP) to perform the double duty of diagnosis and therapy in atherosclerosis treatment regime. A library of dual-targeted NPs with an encapsulated iron oxide NP, mito-magneto (MM), with a magnetic resonance imaging (MRI) contrast enhancement capability was elucidated. Relaxivity measurements revealed that there is a substantial enhancement in transverse relaxivities upon the encapsulation of MM inside the dual-targeted NPs, highlighting the MRI contrast-enhancing ability of these NPs. Successful in vivo imaging documenting the distribution of MM-encapsulated dual-targeted NPs in the heart and aorta in mice ensured the diagnostic potential. The presence of mannose receptor targeting ligands and the optimization of the NP composition facilitated its ability to perform therapeutic duty by targeting the macrophages at the plaque. These dual-targeted NPs with the encapsulated MM were able to show therapeutic potential and did not trigger any toxic immunogenic response.

Nanotechnology-mediated crossing of two impermeable membranes to modulate the stars of the neurovascular unit for neuroprotection.

The success of nanoparticle-mediated delivery of antioxidant and antiinflammatory-based neuroprotectants to the brain to improve neuronal functions in neurodegenerative diseases has demonstrated lesser impact instead of achieving its full potential. We hypothesized that these failures were due to a combination of parameters, such as: (i) unavailability of a delivery vehicle, which can reproducibly and efficiently transport through the brain capillary endothelium; (ii) inefficient uptake of therapeutic nanoparticles in the neuronal cell population; and (iii) limited ability of a single nanoparticle to cross the two most-impermeable biological barriers, the blood-brain barrier and mitochondrial double membrane, so that a nanoparticle can travel through the brain endothelial barrier to the mitochondria of target cells where oxidative damage is localized. Herein, we demonstrate optimization of a biodegradable nanoparticle for efficient brain accumulation and protection of astrocytes from oxidative damage and mitochondrial dysfunctions to enhance the neuroprotection ability of astrocytes toward neurons using neurodegeneration characteristics in SOD1G93A rats. This biodegradable nanomedicine platform with the ability to accumulate in the brain has the potential to bring beneficial effects in neurodegenerative diseases by modulating the stars, astrocytes in the brain, to enhance their neuroprotective actions.

Core hydrophobicity tuning of a self-assembled particle results in efficient lipid reduction and favorable organ distribution.

Atherosclerosis, the deadliest disease in the United States, arises due to the build up of plaques in the arteries as a result of excessive cholesterol deposition and an impaired cholesterol removal process. High density lipoproteins (HDL), popularly known as "good cholesterol", are naturally occurring nano-sized particles that, along with apolipoproteins, are deployed to maintain cholesterol homeostasis in the body. Both cholesterol efflux, from the fat-laden macrophages in the arteries, and intracellular lipid transport, to deliver cholesterol to the mitochondria of liver cells for metabolism, hold key responsibilities to maintain healthy lipid levels inside the body. We designed a library of nine mitochondria targeted polymer-lipid hybrid nanoparticles (NPs), comprised of completely synthetic yet biodegradable components, that are capable of performing HDL-like functions. Using this library, we optimized a superior mitochondria targeted NP candidate, which can show favourable organ distribution, therapeutic potential, and non-toxic properties. Two targeted NP formulations with optimum NP size, zeta potential, and cholesterol binding and release properties were identified. Lipid reduction and anti-oxidative properties of these two NPs demonstrated cholesterol removal ability. In vivo therapeutic evaluation of the targeted-NP formulations in apolipoprotein E knockout (apoE-/-) mice indicated lipid reduction and anti-inflammatory properties compared to non-targeted NPs. This synthetic targeted NP with potential abilities to participate in both extra- and intracellular cholesterol transport might potentiate therapeutic interventions for heart diseases.

Centrifugation-Free Magnetic Isolation of Functional Mitochondria Using Paramagnetic Iron Oxide Nanoparticles.

Subcellular fractionation techniques are essential for cell biology and drug development studies. The emergence of organelle-targeted nanoparticle (NP) platforms necessitates the isolation of target organelles to study drug delivery and activity. Mitochondria-targeted NPs have attracted the attention of researchers around the globe, since mitochondrial dysfunctions can cause a wide range of diseases. Conventional mitochondria isolation methods involve high-speed centrifugation. The problem with high-speed centrifugation-based isolation of NP-loaded mitochondria is that NPs can pellet even if they are not bound to mitochondria. We report development of a mitochondria-targeted paramagnetic iron oxide nanoparticle, Mito-magneto, that enables isolation of mitochondria under the influence of a magnetic field. Isolation of mitochondria using Mito-magneto eliminates artifacts typically associated with centrifugation-based isolation of NP-loaded mitochondria, thus producing intact, pure, and respiration-active mitochondria. © 2017 by John Wiley & Sons, Inc.

Terpyridyl oxovanadium(IV) complexes for DNA crosslinking and mito-targeted photocytotoxicity.

Oxovanadium(IV) complexes [VO(L1/L2)Cl2]n+ (1,2) of (anthracenyl)terpyridine (An-tpy as L1 in 1, n=0) and triphenylphosphonium-appended (anthracenyl)terpyridine (An-tpy-TPP+ as L2 in 2, n=1) were synthesized, characterized and their DNA crosslinking ability, photocytotoxicity in visible light and cellular localization in cancer cells studied. The bromide derivative of 2, viz. [VO(An-tpy-TPP)Br2]Br (3) is structurally characterized. The structure showed trans disposition of two halides in the coordination sphere and the TPP+ unit is a pendant to the terpyridyl ligand. The DNA melting and comet assay studies on the complexes suggest the formation of DNA crosslinks. Complexes 1 and 2 displayed ~10 fold increase in cytotoxicity on exposure to visible light (400-700nm) when compared to those in dark in HeLa and MCF-7 cells. FACScan (Fluorescence Associated Cell Sorter Scan) analysis showed cellular apoptosis when treated with the complex in visible light in comparison to their dark controls. Fluorescence microscopic studies using complex 2 revealed its mitochondrial localization within the cancer cells.

Mito-magneto: a tool for nanoparticle mediated mitochondria isolation.

The field of intracellular organelle targeting using nanoparticle (NP) is mushrooming rapidly. Thus, the area of nanotechnology-enabled targeting of mitochondrion, the cellular powerhouse, for diseases characterized by mitochondrial dysfunctions such as cancer, diseases of the central nervous system, and cardiovascular diseases is also growing at a rapid pace. Optimization of a NP's ability to target the mitochondria requires quantification of the particles in this subcellular organelle and isolation of mitochondria from the cells. Conventional gradient centrifugation used in currently available methods may not be appropriate for NP containing mitochondria isolation as these particles undergo Brownian motion under centrifugal forces yielding irreproducible results. There is only one method for centrifugation-free mitochondria isolation; however, this method requires immunoprecipitation. Thus, a reliable centrifugation and immunoprecipitation free method is urgently needed to support this growing field of nanotechnology-based mitochondria targeting. Here, we report a mitochondria-targeted magnetic NP, Mito-magneto, to avoid centrifugation and immunoprecipitation methods for isolation of functional, respiration active pure mitochondria, which can be used to analyze and quantify mitochondria targeting properties of various NPs as an important tool for the growing field of "mitochondrial nanomedicine".

The Platin-X series: activation, targeting, and delivery.

Anticancer platinum (Pt) complexes have long been considered to be one of the biggest success stories in the history of medicinal inorganic chemistry. Yet there remains the hunt for the "magic bullet" which can satisfy the requirements of an effective chemotherapeutic drug formulation. Pt(iv) complexes are kinetically more inert than the Pt(ii) congeners and offer the opportunity to append additional functional groups/ligands for prodrug activation, tumor targeting, or drug delivery. The ultimate aim of functionalization is to enhance the tumor selective action and attenuate systemic toxicity of the drugs. Moreover, an increase in cellular accumulation to surmount the resistance of the tumor against the drugs is also of paramount importance in drug development and discovery. In this review, we will address the attempts made in our lab to develop Pt(iv) prodrugs that can be activated and delivered using targeted nanotechnology-based delivery platforms.

Photorelease and Cellular Delivery of Mitocurcumin from Its Cytotoxic Cobalt(III) Complex in Visible Light.

Ternary cobalt(III) complexes of curcumin (Hcur) and mitocurcumin [Hmitocur, a dicationic bis(triphenylphosphonium) derivative of curcumin] having a tetradentate phenolate-based ligand (H2L), namely, [Co(cur)(L)] (1) and [Co(mitocur)(L)]Cl2 (2), were prepared and structurally characterized, and their photoinduced cytotoxicity was studied. The diamagnetic cobalt(III) complexes show an irreversible Co(III)-Co(II) redox response and a quasireversible curcuminoid-based reduction near -1.45 and -1.74 V SCE, respectively, in DMF/0.1 M [(n)Bu4N](ClO4). The complexes exhibit a curcumin/mitocurcumin-based absorption band near 420 nm. Complex 1 was structurally characterized by X-ray crystallography. The structure contains the metal in a CoN2O4 distorted octahedral coordination arrangement with curcumin binding to the metal in its enolic form. Binding to cobalt(III) increases the hydrolytic stability of curcumin. Complex 2, having a dicationic curcuminoid, shows significant cellular uptake and photoinduced cytotoxicity compared to its curcumin analogue 1. The dicationic cobalt(III) complex 2 has significantly better cellular uptake and bioactivity than the neutral species 1. Complex 2 with mitochondrial localization releases the mitocurcumin dye upon exposure to visible light (400-700 nm) in human breast cancer MCF-7 cells through photoreduction of cobalt(III) to cobalt(II). Complex 2 displays a remarkable photodynamic therapy (PDT) effect, giving an IC50 value of ∼3.9 µM in visible light (400-700 nm) in MCF-7 cells while being much less toxic in the dark (>50 µM). The released mitocurcumin acts as a phototoxin, generating intracellular reactive oxygen species (ROSs). The overall process leads to light-controlled delivery of a curcuminoid (mitocur) into the tumor cells while the dye alone suffers from hydrolytic instability and poor bioavailability.

Nanotechnology inspired tools for mitochondrial dysfunction related diseases.

Mitochondrial dysfunctions are recognized as major factors for various diseases including cancer, cardiovascular diseases, diabetes, neurological disorders, and a group of diseases so called "mitochondrial dysfunction related diseases". One of the major hurdles to gain therapeutic efficiency in diseases where the targets are located in the mitochondria is the accessibility of the targets in this compartmentalized organelle that imposes barriers toward internalization of ions and molecules. Over the time, different tools and techniques were developed to improve therapeutic index for mitochondria acting drugs. Nanotechnology has unfolded as one of the logical and encouraging tools for delivery of therapeutics in controlled and targeted manner simultaneously reducing side effects from drug overdose. Tailor-made nanomedicine based therapeutics can be an excellent tool in the toolbox for diseases associated with mitochondrial dysfunctions. In this review, we present an extensive coverage of possible therapeutic targets in different compartments of mitochondria for cancer, cardiovascular, and mitochondrial dysfunction related diseases.

New formulation of old aspirin for better delivery.

For better use of cyclooxygenase dependent anti-inflammatory properties and mitochondrial activities of aspirin, new hydrophobic analogues of aspirin were developed and successfully encapsulated in polymeric nanoparticles (NPs). In vivo anti-inflammatory effects of these NPs using a mouse model demonstrated unique properties of an optimized aspirin analogue to inhibit production of pro-inflammatory and enrichment of anti-inflammatory cytokines.

Trans-dichlorooxovandium (IV) complex as a novel photoinducible DNA interstrand crosslinker for cancer therapy.

Although DNA interstrand crosslinking (ICL) agents such as mitomycin C, cisplatin and psoralen serve as potent anticancer drugs, these agents are known to have dose-limiting toxic effects on normal cells. Moreover, tumor resistance to these agents has been reported. Here, we show that trans-dichlorooxovanadium (IV) complex of pyrenyl terpyridine (VDC) is a novel photoinducible DNA crosslinking agent. By a combination of in vitro and ex vivo experiments including plasmid-based assays, we find that VDC forms monoadducts on the DNA and can be activated by UV-A and visible light to generate DNA interstrand crosslinks. VDC efficiently activates Fanconi anemia (FA) pathway of DNA interstrand crosslink repair. Strikingly, photoinduction of VDC induces prolonged activation of cell cycle checkpoint and a high degree of cell death in homologous recombination (HR)/ICL repair defective cells. Moreover, VDC specifically targets cells that express pathological RAD51C mutants. These data imply that VDC can be potentially used for cancer therapy and suggest that tumors arising in patients with gene mutations in FA and HR repair pathway can be specifically targeted by a photoactivatable VDC.

Oxovanadium(IV) complexes of curcumin for cellular imaging and mitochondria targeted photocytotoxicity.

Oxovanadium(IV) complexes [VO(R-tpy)(cur)](ClO4) (1, 2) of curcumin (Hcur) and terpyridine ligands (R-tpy) where R is phenyl (phtpy in 1) or p-triphenylphosphonium methylphenyl bromide (C6H4CH2PPh3Br) (TPP-phtpy in 2) were prepared and characterized and their DNA photocleavage activity, photocytotoxicity and cellular localization in cancer cells (HeLa and MCF-7) were studied. Acetylacetonate (acac) complexes [VO(R-tpy)(acac)](ClO4) of phtpy (3) and TPP-phtpy (4) were prepared and used as the control species. These complexes showed efficient cleavage of pUC19 DNA in visible light of 454 nm and near-IR light of 705 nm. Complexes 1 and 2 showed significant photocytotoxicity in visible light of 400-700 nm. FACS analysis showed sub-G1/G0 phase cell-cycle arrest in cancer cells when treated with 1 and 2 in visible light in comparison with the dark controls. Fluorescence microscopic studies revealed specific localization of the p-triphenylphosphonium complex 2 in the mitochondria of MCF-7 cancer cells whereas no such specificity was observed for complex 1.

Carbohydrate-appended photocytotoxic (imidazophenanthroline)-oxovanadium(IV) complexes for cellular targeting and imaging.

Oxovanadium(IV) complexes [VO(aip)(L)](ClO4)2 (L = phtpy, 1; stpy, 2) and [VO(pyip)(L)](ClO4)2 (L = phtpy, 3; stpy, 4), where aip is 2-(9-anthryl)-1H-imidazo[4,5-f][1,10]phenanthroline, pyip is [2-(1-pyrenyl)-1H-imidazo[4,5-f][1,10]phenanthroline, phtpy is (4'-phenyl)-2,2':6',2''-terpyridine and stpy is (2,2':6',2''-terpyridin-4'-oxy)ethyl-ß-D-glucopyranoside, were prepared, characterized and their DNA binding and photocleavage activity, cellular uptake and photocytotoxicity in visible light were studied. The complexes are avid binders to calf thymus DNA (K(b) ~10(5) mol(-1)). They efficiently cleave pUC19 DNA in red light of 705 nm via the formation of HO˙ species. The glucose appended complexes 2 and 4 showed higher photocytotoxicity in HeLa and Hep G2 cells over the normal HEK 293T cells. No such preference was observed for the phtpy complexes 1 and 3. No significant difference in IC50 values was observed for the HEK 293T cells. Cell cycle analysis showed that the glucose appended complexes 2 and 4 are more photocytotoxic in cancer cells than in normal cells. Fluorescence microscopy was done to study the cellular localization of complex 4 having a pendant pyrenyl group.

Enhancing the photocytotoxic potential of curcumin on terpyridyl lanthanide(III) complex formation.

Lanthanide(III) complexes [Ln(R-tpy)(cur)(NO3)2] (Ln = La(III) in 1, 2; Gd(III) in 5, 6) and [Ln(R-tpy)(scur)(NO3)2] (Ln = La(III) in 3, 4; Gd(III) in 7, 8), where R-tpy is 4'-phenyl-2,2':6',2''-terpyridine (ph-tpy in 1, 3, 5, 7), 4'-(1-pyrenyl)-2,2':6',2''-terpyridine (py-tpy in 2, 4, 6, 8), Hcur is curcumin (in 1, 2, 5, 6) and Hscur is diglucosylcurcumin (in 3, 4, 7, 8), were prepared and their DNA photocleavage activity and photocytotoxicity studied. Complexes [La(ph-tpy)(cur)(NO3)2] (1) and [Gd(ph-tpy)(cur)(NO3)2] (5) were structurally characterized. The complexes in aqueous-DMF showed an absorption band near 430 nm and an emission band near 515 nm when excited at 420 nm. The complexes are moderate binders to calf-thymus DNA. They cleave plasmid supercoiled DNA to its nicked circular form in UV-A (365 nm) and visible light (454 nm) via (1)O2 and ˙OH pathways. The complexes are remarkably photocytotoxic in HeLa cells in visible light (λ = 400­700 nm) and are non-toxic in the dark. FACScan analysis of the HeLa cells treated with 2 and 4 showed cell death via an apoptotic pathway. Nuclear localization of 1­4 is evidenced from confocal imaging on HeLa cells. The hydrolytic instability of curcumin gets significantly reduced upon binding to the lanthanide ions while retaining its photocytotoxic potential.