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Triphenylphosphonium Decorated Liposomes and Dendritic Polymers: Prospective Second Generation Drug Delivery Systems for Targeting Mitochondria Constantinos M. Paleos, Dimitris Tsiourvas, and Zili Sideratou Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00237 • Publication Date (Web): 09 Jun 2016 Downloaded from http://pubs.acs.org on June 11, 2016

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Molecular Pharmaceutics

Triphenylphosphonium Decorated Liposomes and Dendritic Polymers: Prospective Second Generation Drug Delivery Systems for Targeting Mitochondria

Constantinos M. Paleos*,1,2 Dimitris Tsiourvas1 and Zili Sideratou1

1. NCSR “Demokritos”, Institute of Nanosciences and Nanotechnology, 15310 Aghia Paraskevi, Attiki, Greece 2. Regulon SA, 7 Afxentiou st., 17455 Alimos, Attiki, Greece

*Corresponding author: Constantinos M. Paleos, NCSR “Demokritos”, Institute of Nanosciences and Nanotechnology, 15310 Aghia Paraskevi, Attiki, Greece. Tel.: +30-210-6503666, FAX: +30-210-6511766, e-mail address: [email protected]

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Table of Contents Targeting Mitochondria with Triphenylphosphonium Decorated Liposomes and Dendritic Polymers can be a Promising Method for Treating a Diversity of Mitochondrial Diseases.


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Abstract

Targeting specific intracellular organelles has been a biological process of significant interest. Specifically, for mitochondrial targeting conventional liposomal and dendritic polymer nanoparticles were selected to be presented in this mini-perspective. Both types of nanoparticles were decorated on their external surface with triphenylphosphonium cation (TPP), a well-known and effective mitochondrial targeting moiety. Due to their advantageous specificity towards mitochondria, these nanoparticles may be considered as prospective second generation drug delivery systems (DDSs). Functionalized liposomal and dendritic nanoparticles are conveniently prepared and although they are encountering several hurdles on their route from the extracellular environment to the interior of mitochondria, they manage to be accumulated inside them in experiments in vitro. Therefore, the TPP-functionalized nanoparticles presented in this miniperspective can prove effective DDSs and efforts should be continued to obtain results that will trigger further studies including clinical studies and hopefully to effective drugs for mitochondrial diseases. In fact, since these DDSs enter and act at the site where the dysfunction exists, a new medicine subspecialty is emerging, the so-called mitochondrial medicine.

Keywords: liposomes, dendritic polymers, triphenylphosphonium cation, mitochondrial targeting



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Introduction Recently there have been considerable efforts to develop Drug Delivery Systems (DDSs) that target intracellular organelles, including mitochondria, for enhancing their drug delivery efficacy. Excellent reviews have appeared1-8 on the subject which either deal with targeting a diversity of organelles or focus on a specific organelle such as mitochondrion. The field is in an early stage of investigation and significant research effort is needed before entering clinical studies.1-4 The present mini-perspective focuses on mitochondrial targeting by employing on one hand the well-established liposomal carriers and on the other hand the promising dendritic polymer drug delivery systems. Both types of carriers can be conveniently functionalized or multi-functionalized and can also encapsulate a variety of bioactive molecules. The lipophilic triphenylphosphonium cation,9 which is a well-known mitochondrial targeting ligand was conveniently introduced on the external surface of these delivery systems. The properties of the selected nanocarriers are further analyzed in the following paragraphs highlighting among others on the facile molecular engineering that the surface of liposomal and dendritic nanoparticles is subjected to. The structural features of Delocalized Lipophilic Cations (DLCs) targeting groups, including the extensively investigated triphenylphosphonium cation (TPP), share both lipophilic and hydrophilic character and induce transport of molecules, polymers or even molecular assemblies to which they are attached, through plasma and mitochondria membranes, to their final destination which is the accumulation or rather access to mitochondria. They can be translocated through bilayer membranes without requiring a complicated up-take mechanism. The delocalized positive charge of TPP facilitates their mitochondria up-take driven by the large negative potential (∆ψm=150-180 mV) of the mitochondrial membrane.9-12 Specifically, they can be accumulated several hundred-fold within mitochondria as shown pictorially in Figure 1. TPP can be conjugated to a diversity of compounds such as antioxidants,13-16 anticancer agents,17-19


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nucleic acids,20 photosensitizers for photodynamic treatment21,22 or drug loaded delivery systems as liposomes or dendritic polymers. These delivery systems will be discussed in the following chapters of this forum. The accumulation of bioactive compounds to mitochondria is of the outmost importance since serious diseases which are difficult to therapeutically treat such as cancer and neurodegenerative disorders that are associated to mitochondrial dysfunction.

Figure

1.

Uptake

of

a

mitochondria-targeted

nanoparticle

functionalized

with

triphenylphosphonium cation(s).

Liposomes,23,24 spherical vesicles whose lipid bilayers membranes are artificial analogs of cell membranes, are the most extensively investigated DDSs and were for the first time prepared by Bangham and his collaborators25 in 1965. Liposomes are comprised of an aqueous core which is surrounded by one or more lipid membranes. Due to their structural features a diversity of therapeutic compounds and imaging probes can be encapsulated in liposomes. Thus, hydrophilic compounds are encapsulated inside the aqueous core, while lipophilic ones in the lipophilic bilayer. Several methods have been developed for laboratory, pilot and industrial production which permit selected drug loaded formulations to enter pharmaceutical markets.26,27 However, it has to be noted that although liposomal formulations reached from the laboratory to the clinic, the vast number of research papers is not commensurate with the rather limited number of the clinical 5 ACS Paragon Plus Environment


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studies that were performed28-31 and the number of available marketable products. Therefore, upgrading liposomal drug delivery properties is still required which can be achieved by proper lipid selection and molecular engineering of their external surface, for instance by the introduction of appropriate functional moieties.32 Proper selection of constituent lipids affects liposomal stability, transport ability though cell membranes and targeting properties for complementary cell receptors. When the prepared liposomes simultaneously fulfill these advantageous properties their drug delivery efficacy is enhanced. According to the established general strategy for the preparation of efficacious liposomal DDSs, multifunctional liposomes have been developed by decorating their external surface with targeting ligands, such as the folate group,33 in order to achieve cell specificity through the ability of these ligands to interact and bind to complementary cell receptors via molecular recognition. In addition, various other groups have been introduced on liposomes’ surface, such as poly(ethylene glycol) that increases the circulation time of liposomes in biological fluids,34,35 penetrating peptides which facilitate the transport of liposomal nanoparticles through cell membranes,36,37 or imaging modalities that allow determination of their intracellular fate in vitro and in vivo.38 Most importantly, liposomes are biocompatible, biodegradable and nonimmunogenic. Moreover, the large number of targeting groups located on liposomes’ surface results in the amplification of their binding to their complementary cell receptors, known as the “multivalent effect”.39,40 In this mini-perspective, in contrast to conventional targeting achieved through molecular recognition of interacting complementary species,41 targeting of liposomes to mitochondria is exclusively induced by the delocalized lipophilic triphenylphosphonium cation which decorates liposomal surface. Its efficacy as targeting ligand for mitochondria is caused by the high negative membrane potential of the latter, 10-12,42 thus accumulating in mitochondria through cell incubation.


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Concerning dendritic polymers,43-46 which is the other type of DDSs selected to be presented in this work, although they exhibit structural diversity, susceptibility to facile functionalization and effective loading capacity for bioactive compounds through covalent or non-covalent binding,47,48 still remain only promising candidates for drug delivery and they have not been translated into the clinic at a significant extent.49 Now that biodegradable dendritic polymers have been commercially developed, it is even more useful to pursue optimization of these carriers by taking advantage of their favorable molecular features and justifying research funds spent for their investigation. As in the case of liposomes, dendritic polymers were also functionalized with the triphenylphosphonium moieties to render dendritic nanoparticles appropriate for targeting mitochondria. Due to extensive interest to target mitochondria, both for imaging and therapeutic applications, a new medicine subspecialty is developing for treating mitochondria dysfunction, the so-called mitochondrial medicine50,51 In this mini-perspective, selected examples are discussed rather in detail in order to definitively show, that the functionalization of liposomal and dendritic nanoparticles with delocalized lipophilic triphenylphosphonium cations induces their accumulation to mitochondria in vitro. The convenience to effectively functionalize liposomal and dendritic carriers giving positive results in in vitro experiments justifies undertaking preclinical and clinical studies which can finally lead to efficacious therapeutic drugs.

2. Targeting mitochondria with triphenylphosphonium functionalized liposomes Long alkyl chain functionalized TPP derivatives have been prepared and employed for the preparation of TPP decorated liposomes, Figure 2. Mixed liposomes consisting of conventional lipids and a lipid bearing at its distal end the TPP moiety, were prepared employing wellestablished methods. Following liposomal formation, TPP cations are localized on the surface of



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liposomes inducing targeting to mitochondria. It should be noted that for improving liposomes drug delivery efficacy to mitochondria it might be needed to further functionalize these liposomes with selected moieties affording multifunctional derivatives, for instance, by introducing poly(ethylene glycol) chains (PEG) at the external surface of liposomes, a process known as PEGylation.50

Figure 2. Schematic representation a mitochondriotropic phospholipid bilayer membrane decorated with an alkyl chain functionalized TPP.

In this context, a liposomal nano-carrier which is able to target mitochondria was prepared53,54 by attaching a mitochondriotropic moiety on the liposomal surface. Stearyltriphenylphosphonium bromide (STPP), shown below, was synthesized and mixed liposomes consisting of lecithin (PC), cholesterol (Chol) and STPP at a molar ratio PC:Chol:STPP = 65:15:2 were prepared. The so-prepared liposomes bear on their surface TPP moieties appropriate for targeting mitochondria. This is achieved by the stearyl lipophilic chain anchored inside the bilayer while TPP is protruding from the liposomal surface (Figure 2). Their mean diameter was ~130 nm, while the ζ-potential value, as expected, was dependent on the amount of incorporated STPP.


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XP+

STPP It has been shown that BT20 cells incubated with STPP liposomes, labeled with 0.5 mol % Rhodamine-PE, show similar fluorescence localization (Figure 3A) with cells which are stained with MitoTracker Red (Figure 3B), a dye with red-fluorescence that stains mitochondria in live cells. Taking into consideration that the fluorescence probe, Rhodamine-PE, is not linked to STTP but incorporated in the bilayer of STPP liposomes, it can be inferred that, at least partially, the STPP liposomes should be accumulated at or close to mitochondria.54



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Figure 3. Epifluorescence images of BT 20 cells incubated with fluorescent-labeled STPP liposomes (A) or stained with MitoTracker Red (B). (Taken from ref. 53). Following the successful preparation of STPP functionalized liposomal nanoparticles, loading with a drug that can act on a mitochondria dysfunction may potentially improve its efficacy. Thus, a sphingolipid signaling molecule, ceramide, was employed,55 since ceramides are known to participate in a variety of cellular signaling including programmed cell death. It has been hypothesized that delivering this compound to mitochondria could result to enhanced apoptosis compared to non-targeted administration. Furthermore, it was investigated whether the application of mitochondriotropic liposomes, loaded with ceramide, improved its antitumor action in vivo. It was found by animal studies that the bio-distribution of liposomes incorporating ceramide and 3 mol% of a PEGylated lipid is not significantly different compared to non-STPP liposomes and most importantly that the accumulation in the tumor was the same in both cases. However, these ceramide-loaded STPP liposomes significantly inhibited tumor growth rate and improved animal survival. Given that the biodistribution of both types of liposomes is the same, the results suggest that the enhanced activity is attributed to the delivery of ceramide specifically to the mitochondria of cancer cells. In another study, paclitaxel loaded in STPP liposomes exhibited mitochondrial localization and, more importantly, this system was proved cytotoxic towards a cell line resistant to paclitaxel.56 Mitochondrial administration of paclitaxel, a well-known multi-active drug acting directly on mitochondria, can provide a strategy for overcoming drug resistance. In fact, cytotoxicity studies proved that paclitaxel STPP-liposomes showed increased toxicity to Ovcar-3 cells compared to plain paclitaxel-loaded liposomes. Sub-cellular distribution studies employing confocal microscopy indicated that STPP liposomes were effective in changing the localization of paclitaxel improving its accumulation to mitochondria and leading to reduced cell survival towards this cell line that is resistant to paclitaxel. Paclitaxel labeled with Oregon Green showed 10 ACS Paragon Plus Environment

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higher co-localization in mitochondria when it was loaded in STPP liposomes (Figure 4A) than when it was loaded in plain liposomes (Figure 4B). Since experiments also showed that there is no significant difference in the cytotoxicity of paclitaxel-loaded STPP-liposomes and plain paclitaxel-loaded liposomes co-administered with STPP-liposomes, it was proposed that the cell death mechanism was caspace-independent.56 It was, also, concluded that the improved efficacy was attributed both to paclitaxel localization in mitochondria and to the toxicity of STPP liposomes.

Figure 4. Confocal images of cells treated (A) with Rhodamine Red-labeled STPP-liposomes while mitochondria are stained with MitoFluor Green and (B) with paclitaxel which is labeled with Oregon Green while mitochondria are stained with MitoFluor Red. Co-localization is indicated with arrows. Nuclei are stained blue with Hoechst 33342. (Taken from ref. 56).



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The studies presented so far used STPP which, having a single long aliphatic chain, is structurally similar to lysophospholipids. These are known to destabilize liposomal bilayer membranes

leading

to

cytotoxicity.

To

overcome

this

undesirable

property,

triphenylphosphonium functionalized phosphatidylcholine lipids bearing two long aliphatic chains, i.e. dipalmitoyl triphenylphosphonium (DPTPP), dimyristoyl triphenylphosphonium (DMTPP) and dioleoyl triphenylphosphonium (DOTPP), shown below, were synthesized and used for the preparation of mixed liposomes57 employing the well-known thin-film hydration method. All liposomal formulations exhibited mitochondria localization comparable to STPPliposomes.

Incubation of BT-20 cells with STPP-liposomes resulted in a reduction of cell viability dependent on the dose, while DOTPP-, DMTPP- and DPTPP-liposomes did not induce a significant reduction, being close to that of plain-liposomes. Additionally, at liposomal concentration of 2.5 mg mL-1, STPP-liposomes caused cell membrane integrity loss of 35%, in 12 ACS Paragon Plus Environment

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contrast to DOTPP-, DMTPP- and DPTPP-liposomes which behaved as plain liposomes causing no cell membrane integrity loss. Finally, in agreement with the previous experiments of the same laboratory, an evaluation of the effect of the above liposomal formulations on the integrity of mitochondrial membranes of BT-20 cells was performed57 by conducting a modified JC-1 assay. When isolated mitochondria were treated with STPP-liposomes the red fluorescence was significantly reduced which was proposed to be due to the perturbation of the mitochondrial membrane potential. This was not, however, the case with the TPP-phospholipid conjugates. It therefore appears that cytotoxicity of STPP-liposomes was attributed to an effect on mitochondrial membranes. It was, therefore, concluded that TPP-phospholipid conjugates are not toxic as STPP although they maintain the mitochondriotropic character.

3.

Targeting mitochondria with triphenylphosphonium functionalized dendritic

polymers Dendritic polymers exhibit tree-like structures and consist of a diversity of symmetric and non-symmetric polymers (the latter commonly known as hyperbranched polymers), dendrigrafts and dendrons.43-46 Among the above dendritic polymers, dendrons and dendrimers are monodispersed, highly branched symmetric macromolecules having dimensions in the nanometer range. They consist of a focal point, repeating units and a large number of end groups that can be functionalized. Hyperbranched polymers on the other hand are conveniently prepared but they are non-symmetric and polydispersed. Both classes exhibit nano-cavities, where of molecules, including bioactive compounds, can be encapsulated. Alternatively, bioactive compounds can be conjugated to the dendritic polymer, mainly through the formation of chemical bonds with the surface end groups. Non-covalent encapsulation of bioactive molecules inside dendritic polymers vs. covalent conjugation has been intensively investigated47,48 and it



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has been shown that covalent binding of active ingredients is advantageous compared to encapsulation of the drug inside nanocavities. It is exactly the property of dendritic polymers to encapsulate or conjugate molecules of pharmacological interest and the presence of TPP moieties on their surface that induce targeting of these dendritic drug delivery systems with the encapsulated drugs to mitochondria organelles. Also, applying the strategy of molecular engineering58,59 to the external groups of dendritic polymers, elaborated functionalization on their surface occurs improving their drug delivery effectiveness. Additionally, these polymers exhibit the so-called adaptive solubility property,60,61 i.e., their ability to behave either as hydrophobic or hydrophilic polymers depending on the solvent in which they are dissolved. In this manner, their transport through the lipophilic bilayer should be facilitated. Along these lines of employing lipophilic cations as targeting ligands, a novel mitochondria-targeted DDS originating from fifth generation poly(amidoamine) (PAMAM) dendrimer, G5, was synthesized by Torchilin et al.62 through its surface functionalization with the mitochondriotropic triphenylphosphonium ligand. In the first stage a fraction of the cationic amino end-groups of G5 was acetylated affording G5-Ac. Subsequently, the mitochondriatargeting

dendrimer,

G5-Ac-TPP,

was

synthesized

by

reacting

3-

carboxypropyl)triphenylphosphonium bromide with the amino end-groups of G5-Ac. The soobtained dendrimers were labeled with fluorescein isothiocyanate (FITC) for assessing cell accumulation with confocal laser scanning microscopy. HeLa cells incubated with each of the above FITC-labelled dendrimeric derivatives, which were also treated with MitoTracker Deep Red and DNA stain Hoechst 33342, revealed a high degree of co-localization for G5-Ac-TPP in mitochondria (Figure 5, yellow color) compared to the other two derivatives.


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Figure 5. Confocal laser scanning microscopy images of Hela cells treated with the FITClabeled dendrimeric derivatives G5, G5-Ac, or G5-Ac-TPP (green). Mitochondria were stained with MitoTracker Deep Red (red) while the nuclei were stained with DNA stain Hoechst 33342 (blue). The yellow color in the images shown in the last column results from the merging of red and green signals, indicating mitochondrial localization in the case of the TPP functionalized dendrimeric derivative G5-Ac-TPP. (Taken from ref. 62).

For investigating whether surface functionalization with TPP, for achieving mitochondrial targeting, affects cytotoxicity, a cell viability experiment of the three dendrimeric derivatives was conducted in normal mouse fibroblast cell line (NIH-3T3) (Figure 6). The functionalized dendrimeric derivatives exhibited significant less toxicity compared to the starting dendrimer G5. Specifically, both G5-Ac and G5-Ac-TPP showed no significant cytotoxicity at all concentrations tested (up to 20 µΜ) after 24h incubation, while the parent dendrimer was cytotoxic at 10 µM. Also, G5-Ac-TPP is cytotoxic at a concentration of 20 µΜ after 48h incubation, while, for the same incubation time, the parent dendrimer was cytotoxic at all 15 ACS Paragon Plus Environment


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concentrations tested. Thus, multifunctionalization of G5 to a molecularly engineered mitochondria-targeted derivative displays also reduced cytotoxicity towards the cancer cell lines examined. This mitochondria-targeting dendrimer has the potential to be an effective drug delivery system by encapsulating bioactive molecules either in the dendrimeric core or by surface conjugation.

Figure 6. Cytotoxicity of G5, G5-Ac and G5-Ac-TPP towards NIH-3T3 cells after incubation for 24 or 48 h. (Taken from ref. 62).

In line with the previous publication and employing the same parent dendrimer, a very recent investigation63 reported the synthesis of a number of TPP functionalized derivatives of the


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fourth generation PAMAM dendrimer with amino end groups (G4). A number of TPP moieties were either covalently linked directly at the amino end-groups (G4-TPP) or at the end of PEG chains that were attached to the dendrimer’s surface (G4-PEGTPP). Their cellular uptake and cytotoxicity was studied in vitro using the A549 cell line, model of the human alveolar carcinoma. The potency of the above compounds to target the mitochondria of A549 cells was studied employing confocal laser scanning microscopy. As in the previous experiments, the dendrimeric derivatives were labeled with FITC, while mitochondria were stained with MitoTracker Deep Red FM. Dendrimers with ~ 5 TPP moieties attached to their surface were able to cross the cell membrane and to accumulate in mitochondria as proved both by confocal laser scanning microscopy and fluorescence activated cell sorting (FACS), Figure 7. Accumulation in mitochondria was further enhanced in the derivative bearing ~ 10 TPP moieties per dendrimer. However, this derivative was significantly cytotoxic. For G4-PEGTPP derivatives, the presence of the PEG chains reduced their cytotoxicity without significantly compromising their mitochondriotropic ability. The results obtained clearly show that G4-TPP derivatives have the potential and should be further studied as a DDS able to target mitochondria.



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Figure 7. Confocal laser scanning microscopy images of A549 cells treated with FITC-labeled (green) dendrimeric derivatives G4-TPP (having 5 or 10 TPP moieties per dendrimer) or G4PEG-TPP (having 5,10 or 21 PEG-TPP moieties per dendrimer). Mitochondria were stained with MitoTracker Deep Red FM (red) while the nuclei were stained with DNA stain Hoechst 33342 (blue). The yellow color in the images shown in the last column results from the merging of red and green signals, indicating mitochondrial localization of the TPP functionalized dendrimeric derivatives. (Taken from ref. 63).

Within the framework of targeting bioactive compounds to mitochondria, the TPP moiety attached on a lipophilic alkyl chain was introduced to oligo(L-lysine), P1, affording the dendritic carrier P1-TPP.64 D-luciferin, which is the substrate of the firefly enzyme luciferace, was also attached to this carrier. The localization of the parent fluorescein labeled oligomer (P1-FITC) and of its functionalized counterpart with TPP (P1-TPP-FITC) was assessed by employing DU145 human prostate cells. The results shown in Figure 8 provide evidence that 18 ACS Paragon Plus Environment

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functionalization of P1 with alkyl TTP groups leads to effective cell internalization, and specifically to mitochondria. Furthermore, when D-luciferin (luc) was covalently bound to P1TPP it was found that it still acts as an active substrate for luciferase. Confocal microscopy images (Figure 8) have shown that P1-PPT-luc-FITC is localized in the mitochondria of DU145 cells.

Figure 8. Confocal laser scanning microscopy images of DU145 cells treated with the FITClabeled oligo(lysine) derivative P1-TPP-luc-FITC (left image, green). Mitochondria were stained with MitoTracker Red (middle image, red). The yellow color in the overlay image (right) results from the merging of red and green signals, indicating mitochondrial localization of the TPP functionalized oligo(lysine) derivative. (Taken from ref. 64). 19 ACS Paragon Plus Environment


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In an analogous strategy, hyperbranched poly(ethyleneimine), PEI, was functionalized with TPP affording PEI-TPP.65 This polymer is insoluble in water but has the property to form nanoparticles having diameter of ~ 100 nm in phosphate buffer saline or OPTIMEM. To these nanoparticles, Doxorubicin (DOX), a known anticancer drug, was encapsulated (PEI-TPP-DOX) and administered to DU145 human prostate carcinoma cells. Among other results, including the triggering of rapid and severe cytotoxicity, the critical one is that the polymer is localized specifically in mitochondria as indicated by monitoring DOX fluorescence in confocal microscopy experiments. It should be noted that DOX is typically located in the nuclei when administered alone. As shown in Figure 9 DOX (red color) co-localizes with the mitochondrial probe MitoTracker Green FM (green color). This demonstrates the high specificity of the above dendritic nanocarrier to target mitochondria even when considerably low DOX concentration (250 nM) was utilized.

Figure 9. Confocal laser scanning microscopy images of DU145 cells incubated with MitoTracker Green FM (left image, green) and with PEI-TPP-DOX (middle image, red). The yellow color in the overlay image (right) results from the merging of red and green signals, indicating

mitochondrial

localization

of

DOX

encapsulating

TPP

functionalized

poly(ethyleneimine). (Taken from ref. 65). 20 ACS Paragon Plus Environment

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The cytotoxicity of the carrier PEI-TPP as well as the toxicity of DOX encapsulated in this carrier (PEI-TPP-DOX) was assessed and compared with the respective cytotoxicity of free DOX towards DU145 cells. It was found (Figure 10) that the toxicity of the carrier was concentration dependent, but in all cases was relatively low showing a maximum cytotoxicity of 25 % (cell survival 75%) when assayed at 72 h post-incubation and at the maximum concentration tested. The toxicity of free DOX was also concentration dependent having a maximum cytotoxicity of 75% at a concentration of 5 µΜ. On the other hand, DOX encapsulated in the carrier is highly toxic towards DU145 cells at all concentrations employed, even at the lowest one tested (1 µΜ). It is evident that delivering this drug directly to cell mitochondria significantly increases its anticancer activity. In addition, it was found that the mode of cell death after DOX administration changes from slow apoptotic, in the case of free-DOX, to rapid necrotic.65

Figure 10. Cytotoxicity of PEI-TPP, PEI-TPP-DOX and free DOX towards DU145 cells at 24, 48 and 72 h post-incubation. (Taken from ref. 65).



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4. Concluding Remarks and Outlook Functionalization of liposomes and dendritic polymers with triphenylphosphonium cations led to the preparation of drug carriers which effectively target cell’s mitochondria as proved primarily with in vitro experiments. They are relatively stable, remaining intact until their final destination, i.e. during their route from extracellular space to mitochondria by transporting plasma and mitochondria membranes. These “precision” nanoparticles8 targeting specific organelles can also be characterized as prospective second generation Drug Delivery Systems. Furthermore, since mitochondrial dysfunction is associated linked to aging and a diversity of diseases, including, among others, cancer, cardiovascular, and neurodegenerative diseases, and given that the described in this work in vitro experiments were promising, interest should be raised in applying this strategy51,66,67 to detailed and quantitative preclinical and clinical studies. Only upon the conclusion of these experiments, proof will be provided which of the above formulations are effective for treating mitochondrial diseases.


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References (1) Maity, A. R.; Stepensky, D. Limited Efficiency of Drug Delivery to Specific Intracellular Organelles Using Subcellularly “Targeted” Drug Delivery Systems”, Mol. Pharmaceutics 2016, 13, 1-7. (2) Maity, A. R.; Stepensky, D. Delivery of Drugs to Intracellular Organelles Using Drug Delivery Systems: Analysis of Research Trends And Targeting Efficiencies. Int. J. Pharm. 2015, 496, 268-74. (3) Durazo, S. A.; Kompella, U. B. Functionalized Nanosystems for Targeted Mitochondrial Delivery, Mitochondrion 2011, 12, 190-201. (4) Huang, J. G.; Leshuk, T.; Gu, F. X. Emerging Nanomaterials for Targeting Subcellular Organelles. Nano Today 2011, 6, 478-492. (5) Weissig V. From Serendipity to Mitochondria-Targeted Nanocarriers. Pharm. Res. 2011, 28, 2657-2668. (6) Biswas, S.; Torchilin, V. P. Nanopreparations for Organelle-Specific Delivery in Cancer. Adv. Drug Delivery Rev. 2014, 66, 26-41. (7) Pathak, R. K.; Kolishetti, N.; Dhar, S. Targeted Nanoparticles in Mitochondrial Medicine. WIRES Nanomed Nanobiotechnol 2014, doi: 10.1002/wnan.1305. (8) Wen, R.; Banik, B.; Pathak, K. P.; Kumar, A. ;Kalishetti, N; Dhar, S. Nanotechnology Inpired Tools for Mitochondrial Dyssfunction Related Diseases. Advanced Drug Delivery Reviews, In Press. (9) Ross, M. F.; Kelso, G. F.; Blaikie, F. M.; James, A. M.; Cochemé, H. M.; Filipovska, A.; Da Ros, T.; Hurd, T. R.; Smith, R. A. J.; Murphy, M. P. Lipophilic Triphenylphosphonium Cations as Tools in Mitochondrial Bioenergetics And Free Radical Biology. Biochemistry (Moscow) 2005, 70, 222-230.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 30

(10) Finichiu, P. G.; James, A. M.; Larsen, L.; Smith, R. A. J.; Murphy, M. P. Mitochondrial Accumulation of a Lipophilic Cation Conjugated to an Ionisable Group Depends on Membrane Potential, pH Gradient And pka: Implications for the Design of Mitochondrial Probes And Therapies. J. Bioenerg. Biomembr. 2013, 45, 165-173. (11) Hoye, A. T.; Davoren, J. E.; Wipf, P.; Fink, M. P.; Kagan, V. E. Targeting Mitochondria. Acc. Chem. Res. 2008, 41, 87-97. (12) Porteous, C. M.; Logan, A.; Evans, C.; Ledgerwood, E. C.; Menon, D. K.; Aigbirhio, F.; Smith, R. A. J.; Murphy, M. P. Rapid Uptake of Lipophilic Triphenylphosphonium Cations by Mitochondria In Vivo Following Intravenous Injection: Implications for Mitochondria-Specific Therapies And Probes. Biochim. Biophys. Acta 2010, 1800, 1009-1017. (13) Murphy, M. P.; Smith, R. A. J. Targeting Antioxidants to Mitochondria by Conjugation to Lipophilic Cations. Annu. Rev. Pharmacol. Toxicol. 2007, 47, 629-656. (14) Sharma, A.; Soliman, G. M.; Al-Hajaj, N.; Sharma, R.; Maysinger, D.; Kakkar, A. Design And Evaluation of Multifunctional Nanocarriers for Selective Delivery of Coenzyme Q10 to Mitochondria. Biomacromolecules 2012, 13, 239-252. (15) Adlam, V. J.; Harrison, J. C.; Porteous, C. M.; James, A. M.; Smith, R. A. J.; Murphy, M. P.; Sammut, I. A. Targeting an Antioxidant to Mitochondria Decreases Cardiac IschemiaReperfusion Injury. FASEB J. 2005, 19, 1088-1095. (16) Jin, H.; Kanthasamy, A.; Ghosh, A.; Anantharam, V.; Kalyanaraman, B.; Kanthasamy, A. G. Mitochondria-Targeted Antioxidants for Treatment of Parkinson’s Disease; Preclinical And Clinical Outcomes. Biochim Biophys Acta 2014, 1842, 1282-1294. (17) Biswas, S.; Dodwadkar, N. S.; Deshpande, P. P.; Torchilin, V. P. Liposomes Loaded With Paclitaxel And Modified With Novel Triphenylphosphonium-PEG-PE Conjugate Possess Low Toxicity, Target Mitochondria And Demonstrate Enhanced Antitumor Effects In Vitro And In Vivo. J. Controlled Release 2012, 159, 393-402.


Page 25 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18) Han, M.; Vakili, M. R.; Soleymani Abyaneh, H.; Molavi, O.; Lai, R.; Lavasanifar, A. Mitochondrial Delivery of Doxorubicin via Triphenylphosphine Modification for Overcoming Drug Resistance in MDA-MB-435/DOX Cells. Mol. Pharmaceutics 2014, 11, 2640-2649. (19) Zhou, J.; Zhao, W. Y.; Ma, X.; Ju, R. J.; Li, X. Y.; Li, N.; Sun, M. G.; Shi, J. F.; Zhang, C. X.; Lu, W. L. The Anticancer Efficacy of Paclitaxel Liposomes Modified with Mitochondrial Targeting Conjugate in Resistant Lung Cancer. Biomaterials 2013, 34, 3626-3638. (20) Wang, X.; Shao, N.; Zhang, Q.; Cheng, Y. Mitochondrial Targeting Dendrimer Allows Efficient And Safe Gene Delivery. J. Mater. Chem. B 2014, 2, 2546 -2553. (21) Zhang, C.-J., Hu, Q.; Feng, G.; Zhang, R.; Yuan, Y.; Lu, X.; Liu, B. Image-Guided Combination Chemotherapy And Photodynamic Therapy Using a Mitochondria-Targeted Molecular Probe with Aggregation-Induced Emission Characteristics. Chem. Sci. 2015, 6, 45804586. (22) Lei, W.; Xie, J.; Hou, Y.; Jiang, G.; Zhang, H.; Wang, P.; Wang, X.; Zhang, B. Mitochondria-Targeting Properties And Photodynamic Activities of Porphyrin Derivatives Bearing Cationic Pendant. J. Photochem. Photobiol., B: Biology 2010 98, 167-171. (23) Allen, Τ. M.; Cullis, P. R. Liposomal Drug Delivery Systems: From Concept to Clinical Applications. Adv. Drug Delivery Rev. 2013, 65, 36-48. (24) Pattni, B. S.; Chupin, V. V.; Torchilin, V. P. New Developments in Liposomal Drug Delivery. Chem. Rev. 2015, 115, 10938–10966. (25) Bangham, A. D.; Standish, M. M.; Watkins, J. C. Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids. J. Mol. Biol. 1965, 13, 238-252. (26) Wagner, A.; Vorauer-Uhl, K. Liposomes Technology for Industrial Purposes. J. Drug Delivery 2011, doi:10.1155/2011/591325. (27) Arias, J. L. Liposomes in Drug Delivery: A Patent Review (2007-present). Expert Opin. Ther. Pat. 2013, 23, 1399-1414.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 30

(28) Dua, J. S.; Rana, A. C.; Bhandari, A. K. Liposome: Methods of Preparation And Applications. Int. J. Pharm. Studies and Research 2012, III, 14-20. (29) Sen, K.; Mandal, M. Second Generation Liposomal Cancer Therapeutics: Transition From Laboratory to Clinic. Int. J. Pharm. 2013, 448, 28-43. (30) Venditto, V. J.; Szoka Jr, F. C. Cancer Nanomedicines: so Many Papers And so Few Drugs! Adv. Drug Delivery Rev. 2013, 65, 80-88. (31) Kraft, J. C.; Freeling, J. P.; Wang, Z.; Ho, R. J. Emerging Research And Clinical Development Trends of Liposome And Lipid Nanoparticle Drug Delivery Systems. J. Pharm. Sci. 2014, 103, 29-52. (32) Torchilin, V. P. Multifunctional And Stimuli-Sensitive Pharmaceutical Nanocarriers. Eur. J. Pharm. Biopharm. 2009, 71, 431-444. (33) Gabizon, A.; Horowitz, A. T.; Goren, D.; Tzemach, D.; Mandelbaum-Shavit, F.; Qazen, M. M.; Zalipsky, S. Targeting Folate Receptor With Folate Linked to Extremities of Poly(Ethylene Glycol)-Grafted Liposomes: In Vitro Studies. Bioconjugate Chem. 1999, 10, 289-298. (34) Lasic, D. D.; Needham, D. The "Stealth" Liposome: A Prototypical Biomaterial. Chem. Rev., 1995, 95, 2601-2628 and references cited therein. (35) Silvander, M.; Hansson, P.; Edwards, K. Liposomal Surface Potential And Bilayer Packing as Affected by PEG-Lipid Inclusion. Langmuir, 2000, 16, 3696-3702. (36) Tseng, Y. L.; Liu, J. J.; Hong, R. L. Translocation of Liposomes into Cancer Cells by CellPenetrating Peptides Penetratin And Tat: A Kinetic And Efficacy Study. Mol. Pharmacol. 2002, 62, 864-872. (37) Torchilin, V. P. Cell Penetrating Peptide-Modified Pharmaceutical Nanocarriers for Intracellular Drug And Gene Delivery. Biopolymers, 2008, 90, 604-610. (38) Janib, S. M.; Moses, A. S.; MacKay, J. A. Imaging And Drug Delivery Using Theranostic Nanoparticles. Adv. Drug Delivery Rev. 2010, 62, 1052-1063.


Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(39) Mammen, M.; Choi, S.-K.; Whitesides, G.M. Polyvalent Interactions in Biological Systems: Implications for Design And Use of Multivalent Ligands and Inhibitors. Angew. Chem., Int. Ed., 1998, 37, 2755-2794. (40) Galeazzi, S.; Hermans, T. M.; Paolino, M.; Anzini, M.; Mennuni, L.; Giordani, A.; Caselli, G.; Makovec, F.; Meijer, E. W.; Vomero, S.; Cappelli, A. Multivalent Supramolecular Dendrimer-Based Drugs. Biomacromolecules, 2010, 11, 182-186. (41) Paleos, C. M.; Pantos, A. Molecular Recognition and Organizational and Polyvalent Effects in Vesicles Induce the Formation of Artificial Multicompartment Cells as Model Systems of Eukaryotes. Acc. Chem. Res. 2014, 47, 1475–1482. (42) Modica-Napolitano, J. S.; Aprille, J. R. Delocalized Lipophilic Cations Selectively Target the Mitochondria of Carcinoma Cells. Adv. Drug Delivery Rev. 2001, 49, 63-70. (43) Svenson, S.; Tomalia, D. A. Dendrimers in Biomedical Applications - Reflections on the Field. Adv. Drug Delivery Rev. 2005, 57, 2106-2129. (44) Newkome, G. R.; Shreiner, C. D. Poly(amidoamine), Polypropylenimine, And Related Dendrimers And Dendrons Possessing Different 1→2 Branching Motifs: An Overview of the Divergent Procedures. Polymer 2008, 49, 1-173. (45) Khandare, J.; Calderón, M.; Dagia, N. M.; Haag, R. Multifunctional Dendritic Polymers in Nanomedicine: Opportunities And Challenges. Chem. Soc. Rev. 2012, 41, 2824-2848. (46) Nanjwade, B. K.; Bechra, H. M.; Derkar, G. K.; Manvi, F. V.; Nanjwade, V. K. Dendrimers: Emerging Polymers for Drug-Delivery Systems. Eur. J. Pharm. Sci. 2009, 18, 185196. (47) Kaminskas, L. M.; McLeod, V. M.; Porter, C. J. H.; Boyd, B. J. Association of Chemotherapeutic Drugs with Dendrimer Nanocarriers: An Assessment of the Merits of Covalent Conjugation Compared to Noncovalent Encapsulation. Mol. Pharm. 2012, 9, 355-373.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 30

(48) Kazzouli, S. E.; Mignani, S.; Bousmina, M.; Majoral, J.-P. Dendrimer Therapeutics: Covalent and Ionic Attachments. New J. Chem. 2012, 36, 227-240. (49) Svenson, S. The Dendrimer Paradox – High Medical Expectations But Poor Clinical Translation. Chem. Soc. Rev. 2015, 44, 4131-4144. (50) Armstrong, J. S. Mitochondrial Medicine: Pharmacological Targeting of Mitochondria in Disease. Br. J. Pharmacol. 2007, 151, 1154-1165. (51) Edeas, M.; Weissig, V. Targeting Mitochondria: Strategies, Innovations And Challenges: The Future of Medicine Will Come Through Mitochondria. Mitochondrion 2013, 13, 389-390. (52) Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S. Poly(ethylene glycol) in Drug Delivery: Pros and Cons as well as Potential Alternatives. Angew. Chem. Int. Ed. Engl. 2010, 49, 6288-6308. (53) Boddapati, S. V.; Tongcharoensirikul, P.; Hanson, R. N.; D'Souza, G. G. M.; Torchilin, V. P.; Weissig, V. Mitochondriotropic Liposomes. J. Liposome Res. 2005, 15, 49-58. (54) Weissig, V.; Boddapati, S. V.; Cheng, S.-M.; D'Souza, G. G. M. Liposomes And LiposomeLike Vesicles for Drug And DNA Delivery to Mitochondria. J. Liposome Res. 2006, 16, 249264. (55) Boddapati, S. V.; D'Souza, G. G. M.; Erdogan, S.; Torchilin, V. P.; Weissig, V. OrganelleTargeted Nanocarriers: Specific Delivery of Liposomal Ceramide to Mitochondria Enhances Its Cytotoxicity In Vitro And In Vivo. Nano Lett. 2008, 8, 2559–2563. (56) Solomon, M. A.; Shah, A. A.; D’Souza, G. G. M. In Vitro Assessment of the Utility of Stearyl Triphenyl phosphonium Modified Liposomes in Overcoming the Resistance of Ovarian Carcinoma Ovcar-3 Cells to Paclitaxel. Mitochondrion 2013, 13, 464-472. (57) Benien, P.; Solomon, M. A.; Nguyen, P.; Sheehan, E. M.; Mehanna, A. S.; D'Souza, G. G. M.

Hydrophobized

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Mitochondriotropic Liposomes: Choice of Hydrophobic Anchor Influences Cytotoxicity but not Mitochondriotropic Effect. J Liposome Res. 2016, 26, 21-27. (58) Paleos, C. M.; Tsiourvas, D.; Sideratou, Z. Molecular Engineering of Dendritic Polymers And Their Application as Drug And Gene Delivery Systems. Mol. Pharmaceutics 2007, 4, 169188. (59) Paleos, C. M.; Tsiourvas, D.; Sideratou, Z.; Tziveleka, L.-A. Drug Delivery Employing Mu1ltifunctional Dendrimers And Hyperbranched Polymers. Expert Opin. Drug Deliv. 2010, 7, 1387-1398. (60) Rothland, J. B.; Jessop, T. C.; Wender, P. A. Adaptive Translocation: The Role of Hydrogen Bonding And Membrane Potential in the Uptake of Guanidinium-Rich Transporters into Cells. Adv. Drug Delivery Rev. 2005, 57, 495-504. (61) Tsogas, I.; Tsiourvas, D.; Nounesis, G.; Paleos, C. M. Modelling Cell Membrane Transport: Interaction of Guanidinylated Poly(Propylene Imine) Dendrimers with a Liposomal Membrane Consisting of Phosphate Based Lipids. Langmuir 2006, 22, 11322-11328. (62) Biswas, S.; Dodwadkar, N. S.; Piroyan, A.; Torchilin, V. P. Surface Conjugation of Triphenylphosphonium to Target Poly(amidoamine) Dendrimers to Mitochondria. Biomaterials 2012, 33, 4773-4782. (63) Bielski, E. R.; Zhong, Q.; Brown, M.; da Rocha, S. R. P. Effect of the Conjugation Density of Triphenylphosphonium Cation on the Mitochondrial Targeting of Poly(amidoamine) Dendrimers. Mol. Pharmaceutics 2015, 12, 3043–3053. (64) T.A. Theodossiou, Z. Sideratou, D. Tsiourvas, C.M. Paleos, A Novel Mitotropic Oligolysine Nanocarrier: Targeted Delivery of Covalently Bound D-Luciferin to Cell Mitochondria. Mitochondrion 2011, 11, 982-986.



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Page 30 of 30

(65) Theodossiou, T. A.; Sideratou, Z.; Katsarou, M. E.; Tsiourvas, D. Mitochondrial Delivery of Doxorubicin by Triphenylphosphonium-Functionalized Hyperbranched Nanocarriers Results in Rapid And Severe Cytotoxicity. Pharm. Res. 2013, 30, 2832-2842. (66) Weissig, V.; Cheng, S. M.; D'Souza G. G. M. Mitochondrial Pharmaceutics. Mitochondrion 2004, 3, 229-244. (67) Frantz, M.-C.; Wipf, P. Mitochondria as a Target in Treatment. Environ. Mol. Mutagen. 2010, 51, 462–475.


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