Biomaterials and Bioengineering Approaches for Mitochondria and

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Biomaterials and Bioengineering Approaches for Mitochondria and Nuclear Targeting Drug Delivery Md Nurunnabi, Zehedina Khatun, Abu Zayed Md Badruddoza, Jason R. McCarthy, Yong-kyu Lee, and Kang Moo Huh ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01615 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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ACS Biomaterials Science & Engineering

Biomaterials and Bioengineering Approaches for Mitochondria and Nuclear Targeting Drug Delivery Md Nurunnabia’1, Zehedina Khatunb, Abu Zayed Md Badruddozac, Jason R. McCarthya, Yongkyu Leed,*, Kang Moo Huhe,* a Center

for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston,

MA 02129 United States b Molecular

Cardiology Research Institute, Tufts Medical Center, Boston, MA 02111

United States c Department

of Chemical and Life Science Engineering, Virginia Commonwealth University,

Richmond, VA 23219 United States d Department

of Chemical and Biological Engineering, Korea National University of

Transportation, Chungju 380-706, Republic of Korea e Department

of Polymer Science and Engineering, Chungnam National University, Daejeon

305-764, Republic of Korea 1Current

address: John A. Paulson School of Engineering and Applied Sciences and Wyss

Institute of Biologically Inspired Engineering, Harvard University, 52 Oxford Street, Cambridge, MA 02138, USA

*Corresponding

Authors:

Prof. Yong-kyu Lee, Email: [email protected] Tel: +82-43-841-5224, and Prof. Kang Moo Huh, Email: [email protected], Tel: +82-42-821-7651

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ACS Biomaterials Science & Engineering 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

Contents:

Abstract 1. Introduction 2. Overcoming extracellular matrix within a tissue microenvironment 3. Cell membrane targeting 4. Intracellular targeting 4.1. Mitochondrial targeting 4.1.1. Penetration into the cell as well as the mitochondria 4.1.2. Mitochondrial protein import machinery 4.1.3. Electrostatic force to delocalize the therapeutic 4.2. Nucleus targeting 5. Conclusions and perspectives 6. Acknowledgements 7. References


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ABSTRACT When a therapeutic molecule is administered into a biological system, it faces numerous barriers on its way to the target site. While a portion of the administered dose can overcome these barriers and localize to the targeted site and provide a therapeutic effect, the vast majority either accumulates off-target sites or is excreted without pharmacological benefit. Current drug delivery research aims to address this problem by protecting the therapeutics molecule from the biological milieu so that they can minimize off-target toxicity, while concomitantly overcoming the associated barriers to effectively reach the location of interest. In this review, we focus biomaterials and bioengineering approaches that have potential to overcome the intracellular barriers. We discuss the various systems/ materials developed in so far to deliver drugs and genes and the major challenges that remain unmet. Finally, we provide outlook on importance of subcellular targeting drug delivery and give an overview on potential approaches and materials. Keywords: cell targeting; sub-cellular targeting; sub-cellular localization; mitochondrial delivery; nuclear delivery

1. Introduction

A therapeutic agent faces numerous known and unknown barriers in the body when traveling from its site of administration to the site of action when it is given through any conventional routes such as oral, intravenous, intramuscular, subcutaneous, nasal etc. Over the past few decades, many 3 ACS Paragon Plus Environment


barriers have been identified, and various potential approaches have been also proposed and demonstrated to overcome these barriers.1,2 Different strategies such as particulate formation, polymeric conjugation and encapsulation into a vehicle are likely to be very promising.3-4-5 In recent years, an increasing number of publications intend to focus on developing and understanding various novel techniques for sub-cellular targeting drug delivery and some partial reviews on these have been published. Among them are: intracellular delivery of therapeutic by disrupting the cellular membrane reviewed by Stewart et al.,6 intracellular delivery of antibodies via protein-transduction, liposome, polymer vesicle and viral envelop reviewed by Slastnikova et al.,7 on need of chemical carriers for intracellular delivery of large molecules such as protein and peptide reviewed by Bolhassani A. et al.,8 potential applications of miniatured electroporation for intracellular delivery by Shi et al.,9 and use of bacterial pathogen for sub-cellular drug delivery.10 These strategies are shown to enhance the retention and circulation time as well as modulate halflife and cellular uptake profiles of small-molecule drugs by controlling their aggregation behavior.11 For example, surface decoration or modification with anionic polymers can significantly reduce plasma protein interactions during systemic circulation compared to that of cationic polymeric carriers.12,13,14,15 After leaving the vasculature, the therapeutics are expected to reach the site of action that is typically a particular cell or a group of cells surrounded by the extracellular matrix. To achieve the therapeutic efficacy, an adequate amount of the drug molecules is required to reach the targeting site. Again, in order to reach the therapeutics to the cell, they are required to overcome the barrier of the extracellular matrix that significantly inhibits the molecules from reaching the cell membrane. 16-17 Thus far, a number of potential model systems have been reported, such as incorporating the drugs/gene into a carrier or vehicle that is also modified with affinity ligands (e.g., cell targeting peptides and antibodies) in order to specifically


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localize the drug moieties to the cells of choice.18,19,20 However, as most of the therapeutics usually show activity once inside into the cell, depending on the sites of action, some therapeutics are required to reach cytoplasm, mitochondria or nucleus before achieving a significant therapeutic efficacy. Even then, the therapeutic molecules face barriers inside the cells. Cytoplasmic protein may prevent the molecules from reaching the nucleus or mitochondria. Further, both nuclear and mitochondrial membranes are tight enough to prevent the therapeutics from penetrating into them. An example of thrombin receptor mediated cellular delivery system- Hein et al. developed a delivery approach for targeting proteolytic thrombi, usually over expressed on the outer surface of the cell that plays a unique role for the activation of the G protein.21 They have also mentioned that the same proteolytic thrombi are located in the lysosome. Therefore, proteolytic thrombi targeting mediated drug delivery approach has potential in cytoplasm targeting. In order to help a therapeutic for escaping a lysosomal/endosomal barrier in the intra-cellular environment, research has been focused on developing various polymeric carriers.22,23,24,25 Such polymeric carriers are not only helpful for the small molecules in lysosomal/endosomal escaping but also in increasing retention time in both intra and extracellular environment.26 Anionic polymers shows less interactions with the biomolecules compared to cationic polymers; therefore drug carriers grafted or coated with anionic polymers have longer retention time in systemic circulation. For instance, poly (D, L-lactide-co-glycolide) (PLGA) and polyethylene glycol (PEG) mediated formulations are found to be effective for increasing both retention time and blood circulation half-life.27 Nanoparticulate approach of drug delivery has been emerged as a very promising and comparatively safer approach than that of conventional drug formulations with higher efficacy. In this review, we discuss various approaches and materials that have shown potential for the applications in intracellular targeting drug delivery systems.



2. Overcoming Extracellular Matrix within a Tissue Microenvironment An extracellular matrix is a gel-like soft tissue consisting of cell-secreted molecules that provides structural support around the cells allowing them to connect with each other to form a tissue. The major components of an extracellular matrix are laminin proteins, fibronectin and

Figure 1. The scheme demonstrates the journey of a therapeutic from the site of administration to the site of actions. A certain portion of a non-targeted therapeutics accumulates or deposits on the non-specific site that causes toxicity and side effects in spite of therapeutic effects. Once a therapeutic is administered, a portion of initial administered drug reaches the specific sub-cellular organelles which shows therapeutic effect. The remaining portion that is failed to escape from the biological barriers that occurs during transporting from systemic circulation to organ distribution (A), from organ to tissue (B), from tissue to cells (C) and finally from cell surface to sub-cellular organelles (D). The ultimate therapeutic effect results from the (X=100%-(A+B+C+D)) X amount


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of therapeutics that finally reach to the site of actions after travelling all the way from site of administration.

proteoglycan. The extracellular matrix plays a vital role in protecting cells from the external harsh environment while also connecting the cells each other to form an organ. However, for a therapeutic to reach to the site of action, more specifically the targeted cell, it is required to pass through the barriers of the extracellular matrix around the cells. The extracellular matrix of a tumor tissue contains relatively a higher amount of collagen fibres than those of a normal tissue.28 The difference of compositions in the interstitial fluid results in 2-3 times higher pressure than that of a normal tissue. Therefore, trans-capillary transport of a therapeutic from a blood vessel to a tumor cell faces significant barriers which results in inefficient uptake of therapeutic agents (Figure 2). However, to date many successful nanocarriers have been developed which show potential to overcome the barriers for selectively accumulating in the cancer cells.

Figure 2. (a) Scheme shows the representative rearrangement of cells located in a tissue in association of the extra-cellular matrix. (b) Magnified view of a single cell that is surrounded by 7 ACS Paragon Plus Environment


the extracellular matrix and other cells. A therapeutic agent faces three major barriers such as (i) extracellular matrix, (ii) cell membrane and (iii) endo/lysosomal barrier to reach the nucleus or mitochondria.

Differences in chemical and physiological properties are observed between the extracellular matrix of a normal tissue and a tumor tissue. Several key features of a tumor interstitial compartment (e.g. low proteoglycan, high interstitial fluid pressure and flow, anatomically random-defined lymphatic network, and interstitial convection compared to most normal tissues) favor movement of macromolecules in the tumor interstitium, especially in large tumors.29 In the case of anticancer drug delivery, most investigators have mainly put emphasis on cellular targeting and uptake profile of therapeutics during the design of a formulation but very little attention has been paid on in vivo tumor microenvironment as well as intracellular organelle targeting. The higher pressure in the tumor interstitial fluid compared to that of normal tissue prevents the therapeutic from being able to penetrate into the deep of the tumor. Incorporation of photothermal materials such as gold nanoparticles or graphene oxide have potential for penetrating into the extracellular matrix. The photothermal materials generate heat upon photoirradiation and the optimal heat results in melting of the matrix around cells. When the measured heat reaches around 50 oC, narrow gaps or passages form across the extracellular matrix which facilitates the transportation of the therapeutics closer to cell membrane.30

3. Cell Membrane Targeting The cell membrane is a lipid-lipid bilayer embedded with proteins that mainly protects the intracellular matrix which includes mitochondria, nucleus, lysosome, cytosol and golgi bodies. The cell membrane also prevents the toxic substances from entering into the cell. However, the 8 ACS Paragon Plus Environment

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therapeutics are required to enter into the intracellular organelles by crossing the cell membrane. Many different mechanisms and processes were reported earlier that facilitate the therapeutics to enter into the cell.31,32 Widdows et al. proposed a computational model that demonstrates the mechanism of transportation through the cell membrane while the molecule is linked with amino acids.33 Whether the process is an active or passive transportation, was not clearly understood. The cellular membrane transportation processes have been categorized in two major classes, such as (a) active transportation and (b) passive transportation. Active transportation is defined as entering the molecules into the cell membrane through binding with a particular protein/receptor present in the membrane of a specific cell. On the other hand, passive transportation is defined as entering a molecule through endocytosis and phagocytosis which is also known as reticuloendothelial system (RES), more specifically mononuclear phagocyte system, which are also known as non-specific. The role and mechanism of RES effect in case of intracellular liposomal targeting drug delivery to solid tumor has been reported in a broad and elaborate review article by Maruyama recently.34 This review focus on intracellular distribution of liposomal drug formulation dependent on size and systemic circulation half-life; particles with size ranging from 100 nm to 200 nm in diameter have higher solid tumor targeting potential EPR effect compared to larger or smaller particles.



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Normal

Normal

Tumor

Tumor

Ligand conjugated nanoparticle

Therapeutic

Therapeutic

Active transportation

Nanoparticle

Passive transportation

Figure 3. The scheme represents the transportation mechanism of therapeutics-based active and passive targeted system as well as transportation variation of the therapeutics in normal and tumor tissues. For active and effective targeting, therapeutics could be incorporated with a suitable targeting ligand such as cell penetrating peptide, cell receptor targeting ligand or antibody that specifically binds with a receptor of cell membrane. For an instance, herceptin® which is an immunoglobulin G antibody (IgG), binds with HER-2 receptors, and folic acid binds with folate receptors.35 There are several methods for linking cell membrane targeting moieties with the therapeutics; the antibody or receptor targeting ligand could chemically bind with the therapeutics,36,37,38 or therapeutics could be loaded into the polymers which are chemically bound with cell membrane targeting ligand/peptide.39,40,41 Active targeting offers significant potential features in higher intracellular distribution of therapeutics accumulating inside the cell that may lead to higher therapeutic efficacy (Figure 3). Most importantly, an active targeting delivery system can significantly reduce non-specific distribution with a greater accumulation of therapeutics to the 10 ACS Paragon Plus Environment

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target-specific cells that results in generating lowest toxicity. There are some humanized antibodies which are highly attractive in terms of selectivity and specificity of cell surface receptors.42 These antibodies are also known for distinguishing tumor cells and normal cells in vivo. Some formulations that are designed and developed based on antibodies have been translated from laboratory to commercialization such as Rituxan®, trastuzumab (Herceptin®), cetuximab (Erbitux®), and bevacizumab (Avastin®).43 However, most recent studies have demonstrated that aptamers have more potential than antibodies for targeting specific molecules due to their higher affinity to cells. Aptamer oligonucleotides or peptides have the ability to bind to a specific target molecule like an antibody, though aptamer could even bind to a molecule as small as 60 Dalton unlike an antibody, which binds with a molecule bigger than 600 Daltons. Aptamers have numerous advantages over antibody due to their small size, ease of production, ability to bind with small molecules, better stability and also ease of modification of structure. A study conducted by Xiang et al. demonstrated that aptamer has superior performance in terms of tumor penetration over antibodies.44 This study also showed that it has significant effects on tumor size reduction and imaging as well as on delivering of imaging contrast agent. Many other studies also have indicated such evidence of efficacy of aptamer over antibody.45,46,47,48 A rationale design and development of a therapeutic in association with receptor mediated ligand leads to a promising therapeutic delivery approach. This approach is considered to regulate the side effects that are commonly generated after a cancer patient undergoes chemotherapy. Transferrin, one of the widely considered ligands for both drug and gene delivery, assists the therapeutic to penetrate the cell membrane through the transferrin receptor.49,50,51,52 These transferrin carriers play a vital role for importing iron into the cell based on intracellular iron concentration. However, this receptor has been used by many researchers for the efficient delivery of therapeutics into the cells where



commercially available transferrin is chosen to incorporate with carrier molecules. For instances, Sahoo et al. reported the targeting delivery of paclitaxel to a human prostate cancer cell line in vitro and a prostate cancer cell line in mice in vivo.50 The formulation was prepared through conjugation of transferrin with PLGA followed by loading of the hydrophobic anticancer drug (paclitaxel) inside the PLGA nanoparticle. It was observed that the transferrin-conjugated nanoparticles were significantly efficient for controlling the tumor growth. The mice of control groups (treated with saline and free paclitaxel) were dead after 40 days of observation while the tumor volume was measured to be 10 times larger than the initial state. However, transferrin conjugated nanoparticles treated mice were survived up to 80 days or more with significant tumor volume reduction. Currently antibodies and cell penetrating peptides are also widely used for designing active targeting formulations. However, although cell-penetrating peptide could penetrate the cell membrane of any cells without any selectivity, it enhances cell accumulation both in normal and cancer cells which remains a concern to be resolved. Based on the profile of drug accumulation, it is very important to design a formulation that could exclusively target cancer cells while minimizing the concentration of therapeutics that may be non-specifically up-taken by normal cells. In this regard, we have to find out the unique receptors that are only expressed in cancer cells as well as the receptor-targeting ligands which could be associated with the therapeutics for active targeting delivery. For example, HER-2 receptor is only over expressed in some selective breast cancer cells such as SK-BR3, MDA-MB231 and NSCLC that could be targeted by monoclonal antibody G such as commercially available Herceptin®.53,54 Folate receptor is another example which is overexpressed in KB and A549 cancer cells which could be targeted by folic acid.55,56,57,58


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Table 1. A list of some but commonly used molecules (peptide, antibody, polysaccharides) and their binding receptors. Molecule Folic acid Herceptin Hyaluronic acid RGD (Arg-Gly-Asp) Single-chain variable fragment (ScFv) Transferrin AntiCD3 antibody αvβ3 Anti-T Cell Receptor antibody αDEC-205 antibody KEX2

Target site/receptor Folate receptor HER-2 receptor CD44 VEGF R2 EGF Transferring receptor Fc receptor Integrin receptor T-cell receptor (TcR) DEC-205

Reference 59 35 60 61 62

63 64 65

4. Intracellular Targeting Intracellular targeting of the therapeutics is defined as delivery of the therapeutics to target the organelles such as cytoplasm, mitochondria and nucleus. Intracellular targeting delivery of either drug molecules or therapeutic biosimilars is a challenging area of research for improving their intracellular localization for getting better therapeutic efficacy. Different therapeutics show activity in different sites of cells. For example, therapeutic nucleotides are required to localize into the nucleus to incorporate the existing DNA whereas some therapeutic drug molecules act at nucleus, mitochondria, cytoplasm, and even on the cell membrane. However, nucleus and mitochondria are the most important targeting sites among the intracellular organelles for delivering therapeutics. The mitochondria produce energy such as adenosine triphosphate (ATP) which supplies adequate power to survive, and the nucleus contains DNAs that maintain genetics codes and play a vital role during cell division. Damaging the mitochondria could kill the diseased cells by inhibiting the production of energy/power. On the other hand, delivering of the therapeutic



DNA to the mitochondria can help to repair the defected DNA of an abnormal cell. The similar approach can be applied to nucleus targeted therapeutic molecules delivery too; damaging nucleus can result cell death and repairing the defected nucleus can result cure. 4.1.

Mitochondrial Targeting

The mitochondria, known as the powerhouse of a cell, is one of the vital organelles, which produces energy through oxidative phosphorylation for the cellular activity. It consists of two membranes namely outer mitochondrial membrane having a large number of pores compared to that of inner mitochondrial membrane. Compared to the outer plasma membrane, inner mitochondrial membrane contains more saturated phospholipids and proteins which make it unique over other membranes. In addition, the mitochondria maintain a stable membrane potential (-180 mV). Therefore, it is highly unlikely a larger molecule or biologic can penetrate the membrane to be accumulated into the mitochondrial core. The physico-chemical properties of the molecules are also largely related to their penetrability of the membrane. Although the mitochondria provides energy for performing cellular activities, it plays a major role in regulating cell death following apoptosis. Along with these properties, the mitochondria regulate the tricaboxylic acid and urea cycle, fatty acid oxidation, amino acid metabolism, calcium


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Figure 4. Chemical conjugation between anticancer drug doxorubicin (DOX) and mitochondria penetrating peptide (MPP). The cell images clearly show evidence of subcellular co-localization of DOX and MPP conjugate whereas DOX only locates into the nucleus.66 Reproduced with permission from ref 66. Copyright 2013 ACS Publications.



homeostasis, and redox signaling. By producing energy it keeps cell alive while the dysfunction of mitochondria generates a large number of life killing and threatening diseases like cancer, neurodegenerative diseases, ageing, Friedreich’s ataxia (due to the defect in mitochondrial iron transport),48 Wilson’s disease (caused by a defect in copper metabolism),67 human deafness dystonia syndrome (resulting from a defect in mitochondrial protein import),68 and ischemia– reperfusion injury (due to oxidative damage to mitochondria). Besides, the mitochondria play a major role towards apoptosis by releasing cytochrome c which induces apoptosis after activation of the caspases following some sequential steps in cell cytoplasm. On the other hand, the disruption of electron transport system and energy metabolism, as well as the change in redox potential in the mitochondria also plays a pivotal role to induce apoptosis.69 Therefore, it is highly desirable to deliver drugs targeting the mitochondria which can eliminate or control the diseases associated with this organelle. Strategies to deliver the therapeutic moieties into the mitochondria have been considered to control the diseases associated with mitochondria. Why should the mitochondria be considered for targeting drug delivery as some drugs already naturally target this organelle to control the diseases associated with it? The rationale behind the mitochondrial targeting drug delivery is to enhance the concentration of the drug up to the therapeutic concentration level while preventing random distribution. For a molecule to enter into the mitochondria, two selective steps must be considered; (i) targeting of the routes through which drugs will be entered into the cell and (ii) the selectivity to mitochondria. In case of drugs that naturally target the mitochondria, consideration of the first step is highly required. On the other hand, for drugs that need to be modified to target the mitochondria both steps are required. For examples, various strategies are applied for clinically approved drug paclitaxel,70 and VP-16,71 which allow more drug molecules to penetrate the cancer


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cells and subsequently towards the mitochondria. Some research groups have attempted to deliver drug molecules through developing various nano-formulations such as micelles, liposomes and vesicle wherein drug molecules have been entrapped. Redox potential and/or pH of the cancer cells can also offer new windows to gain access to the mitochondria. Some other groups have introduced redox sensitive or pH sensitive polymers/ materials or both to their delivery vehicles and obtained better therapeutic responses.72,73,74,75 4.1.1. Penetration into the Cell as Well as the Mitochondria To obtain improved therapeutic efficacy from the drugs targeting mitochondria, it must cross both the cell and mitochondrial membrane. Special transporters are required to enter into the mitochondria as it significantly excludes a wide range of ions and molecules. Though the mitochondria offer many unique characteristics to target it, one of the main challenges of drug molecules/therapeutics is to penetrate the membrane of the mitochondria. Following are some potential strategies that have been reported in literature regarding the delivery of therapeutics into mitochondria. 4.1.2. Mitochondrial Protein Import Machinery The mitochondria contains two types of proteins: nuclear encoded mitochondrial proteins and precursor proteins. 76 Most of the mitochondrial proteins are synthesized as precursor proteins in the cytosol which then penetrate into the mitochondria following post-translational mechanism.76 These precursor proteins are divided into two main classes: one type of precursor proteins carries N-terminal cleavable extensions while the other does not contain this extension.53 These positively charged proteins function as targeting signals that interact with the mitochondrial import receptors and direct the pre-proteins across both outer and inner membranes. The translocase of the outer mitochondrial membrane (translocator outer membrane complex consisting of several pre-protein



receptors and a general import pore) represents the central entry gate for practically all nuclearencoded mitochondrial proteins (Figure 4). Drugs that are protein in nature have to pass two different translocators: translocator outer membrane and translocator inner membrane. The complexes that consist of the cleavable Nterminal extension, allow the nucleus-encoded proteins to translocate into the sub-cellular level.77 Inclusion of this sequence to a drug or drug carrier that can aid the necessary proteins of interest to deliver into the mitochondria can be a potential strategy. 4.1.3. Electrostatic Force to Delocalize the Therapeutic Rendering positive (cationic) charge on the drug carriers into which the therapeutic molecules are directly linked or encapsulated inside is considered so that the drug can pass across both cell and mitochondrial membrane through destabilization of the membrane electrochemical gradient. Different delocalized compounds such as triphenylphosphine (TPP) and its derivatives, cancer cell penetrating peptide and dequalinium are widely used as lipophilic cationic compounds for mitochondrial targeting drug delivery. A study performed by Shareefa et al. showed enhanced accumulation of mitoquinone (MitoQ) and R-tocopherol in the mitochondria after conjugation with the lipophilic triphenylphosphonium cations.78 This is due to electrochemical gradient which generates negative charge in the mitochondrial matrix. Sharma et al. developed a multifunctional nanocarrier system based on ABC miktoarm polymers (A = poly(ethylene glycol (PEG), B = polycaprolactone (PCL), and C = triphenylphosphonium bromide (TPPBr)) which could deliver coenzyme Q10 to the mitochondria in adequate quantities, which is not commonly observed in a traditional delivery system.79 It should be noted that a minimum amount of TPP is required to be incorporated in order to facilitate the delivery of the efficient amount of drugs into the mitochondria.78 Although the introduction of TPP into the delivery system has gained significant


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attraction, it is reported that this approach is not selective for particular mitochondria within an organism.80 On the other hand, TPP is more effective for low molecular weight drugs or drug carriers compared to that of high molecular weights. Recently, Cho et al. reported that di-block TPP showed great potential in terms of mitochondrial uptake; in this case TPP was conjugated with both end of poly(ε-caprolactone) diol.81 The lipophilic conjugates formed self-



A

O

OH

O OH OH

OCH3 O

OH

O

.HCl

O

O

+P

HO

CH3 OH NH2

O

Room temperature OH

DCC, NHS

O OH OH

OCH3 O

OH

O

O

CH3 OH HN

P+

Overlay

Free DOX

TPP-DOX

Free DOX

Doxorubicin Mitochondria Hoechst 33342

TPP-DOX

MDA-MB-435/WT

B

MDA-MB-435/DOX

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

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Figure 5. Intracellular distribution of DOX and TPP−DOX in MDAMB-435/WT and MDA-MB435/DOX cells. Cells were exposed to DOX or TPP−DOX solution at 37 °C for 24 h, and the intracellular mitochondria were stained by MitoTracker Green followed by nucleus staining with Hoechst 33342. The cells were observed under confocal laser scanning microscope. The overlap between the fluorescence of DOX (red) and Hoechst 33342 (blue) appears as pink and shows 20 ACS Paragon Plus Environment

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nuclear distribution of DOX in the cells. The overlap between the fluorescence of DOX (red) and MitoTracker (green) appears as orange and shows distribution of DOX in the mitochondria of cells. Reproduced with permission from ref 82. Copyright 2014 American Chemical Society.

assembled nanoparticles where water-soluble and insoluble DOX was loaded physically. The most interesting finding from this study was that water insoluble form of DOX showed higher loading efficiency and lower mitochondrial uptake compared to that of water-soluble form. The in vitro studies suggested that cellular, nuclear, and mitochondrial uptake of the nanoparticle form were 23 times higher than that of molecular form of DOX. Similarly, in another study by Khatun et al. enhanced mitochondrial uptake and better therapeutic efficacy was also observed, where they developed disulfide linked poly(ethylene glycol)−(TPP)2 (PEG− (ss-TPP)2) conjugates and their DOX-loaded nanoparticles. Dequalinium, a dicationic compound that is able to form vehicle known as dequalinium liposomes through aggregation, was also reported to deliver drugs to mitochondria.83 Jeena and co-authors demonstrated that TPP conjugated phenylalanine dipeptide was significantly accumulated inside the mitochondria of cell, inducing mitochondrial dysfunction via membrane disruption, resulting in apoptosis.84 Vaidya et al. showed the delivery of paclitaxel using a liposomal system prepared with dequalinium followed by conjugation of folic acid that further enhanced the cellular uptake of the developed vector after binding with folate receptors.85 To overcome the multidrug resistance, dequalinium conjugated polyethylene glycol distearoyl phosphatidylethanolamine (DQA-PEG2000-DSPE) was prepared to deliver the polyphenolic anticancer drug resveratrol to the mitochondria.86 Application of this compound was also reported by other research groups delivering an anticancer drug.87 Kelly and co-workers reported a peptide known as mitochondria-penetrating peptide (MPP), a short cationic part of peptide used to deliver



an anticancer drug DOX to the mitochondria through electrostatic interactions.88,89 Chamberlain et al. also utilized this peptide for drug delivery through conjugating with DOX via chemical linkage (Figure 5) and investigated their intracellular distribution.66 HeLa cells co-cultured with free DOX were found to exhibit brightest green fluorescence (comes from DOX) in the nucleus, and comparatively less fluorescence in cytoplasm and mitochondria. However, cells, co-cultured with peptide-conjugated DOX, showed brightest green fluorescence around mitochondria and less intensity in the nucleus. These cell images clearly indicate that the peptide can drive the therapeutics to mitochondria. Another promising advantage of mitochondrial targeting drug delivery is to overcome the multi-drug resistance barrier. Once a drug becomes resistant to a specific cell due to frequent or unregulated administration, the drug might still be effective if being directed to mitochondria instead of nucleus. Zhou et al. published a very interesting report earlier where they prepared a paclitaxel liposome incorporated with D-α-tocopheryl polyethylene glycol 1000 succinate triphenylphosphine conjugate (TPGS1000-TPP) for treating drug-resistant lung cancer.90 For evaluating the therapeutic efficacy in vitro and in vivo they considered anticancer drug resistant lung cancer cell A549/cDDP. They found that the targeting paclitaxel liposomes could significantly enhance the cellular uptake and accumulation into the mitochondria compared to taxol and regular paclitaxel liposomes. Very recently, Han et al. reported another triphenylphosphine (TPP)-conjugated DOX formulation that could significantly overcome the drug resistance in MDA-MB345 cells, which are DOX resistant.82 For achieving the obvious therapeutic activity of the conjugates, both resistant and non-resistant cell lines were considered. In vitro anticancer activity was observed using both free DOX and TPP-conjugated DOX in a head-to-head comparison. Results presented in the report revealed that about 70% and 90% of nonresistant MDA-MB345 cell were killed by free DOX and conjugates, respectively at about 1 µM


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equivalent of DOX concentration. However, in case of resistant MDA-MB345 cell about 30% and 85% were killed by free drug and conjugated drug, respectively at 100 µM equivalent of DOX concentration. The mitochondria targeting liposome also showed significant mitochondrial accumulation in vitro and better therapeutic efficacy compared to that of free paclitaxel in vivo. Ruthenium (II) polypyridyl is another type of mitochondrial targeting complex that has been investigated recently by Liu et al. for intracellular delivery of photodynamic anticancer agent.91

5. Nucleus targeting The nucleus of a cell plays a vital role in both normal and disease state, as most of the diseases, such as genetic or non-genetic disorders, are associated with nucleic acids located into the nucleus. The nucleic acid carries all the profiles of the mother cells while they can be divided into two new cells, thus transferring the genetic codes from one generation to the next one.92-93 Therefore, diseases which are associated with genes such as cancer, diabetes, epilepsy and many more may be carried over to someone whose family has a history of these diseases. To treat these diseases, the delivered therapeutics (either drug molecules or therapeutic genes) are required to localize inside the nucleus so that they could interfere the abnormal DNA of the cells. The most efficient and expected therapeutic efficacy would be realized only if the adequate amount of therapeutic agents is taken up by the cells and localized inside the nucleus. However, the ongoing treatment strategies that may control or regulate the intracellular distribution of the therapeutic molecules have not been designed, in terms of rationale, or much optimized. A better understanding on how the constituents of a formulation undergo intracellular trafficking, that leads to determined locations inside cells, could improve the drug design and delivery.94 The intracellular distributions of existing formulations are random and non-specific, therefore only a



Figure 6. (A) A schematic diagram of the procedures for preparing amine group- and TAT-C6FITC peptide-conjugated MSNs and illustration of transport of DOX@MSNs-TAT across the nuclear membrane. (B) CLSM images of MSNs-TAT with diameters of (1) 25, (2) 50, (3) 67, and (4) 105 nm after incubation with Hela cells for (i) 4, (ii) 8, and (iii) 24 h. Scale bars: 5 μm. (C) CLSM images and (bottom) line-scan profiles of fluorescence intensity for Hela cells incubated for 4 h with (i) free DOX, (ii) DOX@MSNs (25 nm), and (iii) DOX@MSNs-TAT (25 nm). The red fluorescence is from DOX, and the blue fluorescence is from 4,6-diamidino-2-phenylindole (DAPI) used to stain the nuclei. The concentration of DOX was 5 μg mL–1. Scale bars: 5 μm. Panel 24 ACS Paragon Plus Environment

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A-C adapted with permission from ref

95.

Copyright 2014 American Chemical Society. TEA:

Triethylamine, TEOS: Tetraethyl orthosilicate, CTAC: Cetrimonium chloride, APTES: (3Aminopropyl)triethoxysilane. portion of cellular accumulated therapeutics could reach to the target site. To overcome this issue, nucleus targeting moieties such as TAT peptide, RGD, and dexamethasone could be introduced with the therapeutics, which showed significant promise in nuclear targeted based delivery and accumulation.96,97,98,99,100 Besides small molecules or peptides, there have been recent reports on smart polymeric carriers which also show potential for nucleus targeted delivery of therapeutics.101,102,103 For instance, Zhong et al. developed a smart, multicomponent polymeric delivery system that is made up of a backbone based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer and a biforked nuclear transport subunit, one end of which was the therapeutic peptide (H1) and the other end the pH responsive fusion peptide composed of octoarginine and NLS.104 This polymeric system combining the pH-responsive structure and detachable sub-units improved the nucleus targeting efficiency by increasing the drug cellular internalization and accumulation. Bioengineered silk has also been found as a potential candidate for nucleus targeting delivery of therapeutics. A study conducted by Yigit et al. showed that it exhibited more than 4fold higher selectivity to nucleus compared to that of widely used polymer PEG.105 Pan et al. developed TAT peptide conjugated mesoporous silica nanoparticles which was loaded with anticancer drug DOX with high payload.95 These developed nanoparticles enter into the nucleus through importin α and β present in the pore of the nuclear complex as shown in Figure 6. To better understand their optimum size and time of maximum cellular accumulation, the cellular uptake profile of TAT-conjugated silica nanoparticles was investigated. It was reported that the cellular uptake and subcellular distribution is highly dependent on the size of the 25 ACS Paragon Plus Environment


particles.106,107,108,109,110 Particles with diameters of 25 nm and 50 nm show higher uptake and accumulation in cytoplasm and nuclei after 8 and 24 h of incubation respectively compared to 67 and 105 nm particle after 4 h of incubation. Liu et al. demonstrated an advanced design and development of a formulation based on nuclear targeted cancer therapy and real time simultaneous imaging with upconversion nanoprobes.111 The TAT peptide (TAT: YGRKKRRQRRR) was linked with mPEG modified NaGdF4 nanoparticles through thiolation reaction followed by DOX loading. The final hydrodynamic diameter of the nanoparticles was around 60 nm with a positive zeta potential value ranging between 20-30 mV. Due to the cationic surface properties, nanoparticles were highly accumulated inside the cell as well as localized inside the nucleus through the electrostatic interaction. However, since the in vivo studies were not conducted in this study, the biological stability and interaction profile in vivo were not completely understood, although cationic particles are known to be less stable during systemic circulation with less retention time due to aggregation and interactions with biomolecules. It was concluded that the DOX loaded targeted formulation exhibited about 4 fold higher nuclear localization ratio compared to that of free DOX after 24 h of incubation whereas the measured amount of DOX in the cytosole/membrane localization were 2-3 fold higher than that of DOX loaded targeted formulation. It was also demonstrated that particles with size ranging from 25-50 nm had a higher nuclear uptake ratio compared to larger particles which is in agreement with the results reported by Pante et al.,112 where they stated that particles size close to 39 nm in diameter could be translocated into nucleus.


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Figure 7. A schematic illustration of vasculature-to-cell membrane-to-nucleus sequential targeted drug delivery based on RGD and TAT peptides co-conjugated MSNs for effective cancer therapy. Step I: RGD-directed tumor vasculature and cell membrane targeting; Step II: TAT-mediated nuclear targeting. Reproduced with permission from ref 113. Copyright 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Therefore, the anticancer drug DOX was loaded into 25 nm TAT-conjugated silica nanoparticles and co-incubated with HeLa cells to observe the intracellular release of DOX and localization using confocal microscope. Free DOX and DOX loaded silica nanoparticles which were not linked with TAT were considered as positive and negative controls, respectively. Figure 6d shows that the free DOX, randomly distributed across the cells as red fluorescence, is found in the entire cell. In case of TAT-conjugated silica nanoparticles that are loaded with DOX, the fluorescence was found in the nucleus indicating the highest accumulation of the formulation. The opposite scenario was found in case of the nanoparticles that were not conjugated with TAT. These in vitro studies suggested that the specific TAT peptides could enhance the nucleus uptake of therapeutics through active targeting strategy. Another version of charge reversal nanoparticle was reported by Xu et al. for nucleus targeting drug delivery, where they designed and developed polymeric 27 ACS Paragon Plus Environment


nanoparticles comprised of poly(ε-polycaprolactone)-block- polyethyleneimine (PCL-PEI) and folic acid.114 These polymeric particles were pH sensitive, undergoing hydrolysis at acidic pH. The calculated degree of hydrolysis was 75% and 50% at pH 5.0 and 6.0, respectively. Zeta potential values of the polymer nanoparticles were varied based on hydrolysis of amide. For example, zeta potentials were about -20 mV and +50 mV at pH 7.4 and pH 5.0, respectively. Therefore, when the particles accumulate into the cytoplasm, they undergo hydrolysis and thus become highly cationic which enables them to penetrate into the nuclear membrane of the cells. However, these strategies might be considered as non-specific targeting.112 Similar strategies were followed by Zhou et al. for nucleus targeting drug delivery, where they reported that their developed polymeric nanoparticle could hydrolyze at lysosomal pH (5.5) thus change their surface charge from anionic to cationic.115 Positively charged inorganic nanoparticles such as positively charged quantum dots and chitosan nanoparticles were found to show higher selectivity and significant sub-cellular distribution particularly in the nucleus.106,116 Mesoporous nanoparticles have been widely investigated for nucleus targeting drug/gene delivery and were found as an effective strategy of the same.117 Pan et al. designed nanoparticles based on mesoporous silica that efficiently delivered the drug to nucleus and exhibited dramatic tumor regression.113 In this report, they demonstrated that the mesoporous silica nanoparticles decorated with cyclic arginine-glycine-aspartic pentapeptide c(RGDyC) and TAT peptide The formulation specifically binds with angiogenic epithelial cells that are highly overexpressed with integrin αvβ3, but minimally expressed in epithelial cells of normal tissues. This synergistic targeting strategy showed fascinating outcomes in terms of cellular uptake and nuclear localization of the drugs. The RGD/TAT first facilitates them to enter into the


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cell membrane through vasculature cell membrane targeting and then the TAT enhances the nuclear localization (Figure 7).



Figure 8. (A) A schematic of polymer modification, drug encapsulation and disassembly of nanoparticles in response to pH and redox potential. (B) Confocal microscopy images of HCT-116 cells treated with free DOX or DOX encapsulated nanoparticles and line-scale profiles of fluorescence intensity of the cells. Cells were treated for 3 h and fixed with formaldehydes (4.5% in PBS), and the nucleus was stained with Hoechst 33342 (final concentration 1 μg/mL). Cells treated with free DOX and RPDSG/DOX showed concentrated DOX in the nucleus, whereas the cell treated with PDSG/DOX nanoparticles showed an evenly distributed DOX around the nucleus. Note: Nuclei stained with Hoechst 33342 (blue), intracellular DOX (red). Adapted with permission from ref 118 . Copyright 2012 American Chemical Society. GHS: Glutathione.

pH sensitive polymers also play a potential role for nucleus targeting drug and gene delivery as reported by many investigators over the years.119,120,121 For example, Bahadur et al. demonstrated a nuclear targeting drug delivery system based on a peptide conjugated pH sensitive polymer.118 This article reported the formation of a conjugate which is pH sensitive and bioreducible composed of poly(2-(pyridin-2-yldisulfanyl)ethyl acrylate) (PDS) polyethylene glycol and cRGD peptide. Both polyethylene glycol and cRGD peptide are conjugated with PDS polymer through disulfide bonds as shown in Figure 8. Xu et al. has reported on nuclear targeting DNA delivery in association of a pH responsive cationic amphipathic peptide (LAH4-L1) and nuclear localizing signals; Simian virus 40, nucleoplasmin targeting signal, M9 sequence, and the reverse simian virus 40.122 The vehicle was found as an efficient DNA delivery vector that has been able to escape endosome and release the DNA inside the cytoplasm. Zhao et al. has developed a dual responsive (pH/redox) amphiphilic polymer compose of poly(6-O-methacryloyl-d-galactopyranose)-bpoly[2-(diisopropylamino) ethyl methacrylate-co-pyridyl disulfide methylacrylate.123 The


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polymer was further functionalized with folic acid and galactose for hepatoma targeting doxorubicin delivery. The self-assembled micelles that are both acid- and glutathione- sensitive undergo dissociation once they enter into the intracellular environment of a tumor cell. However, nanoparticles were found to be stable in physiological conditions tested in different buffer solutions (such as pH 5.5 and 7.4) with and without glutathione. The conjugated peptide further assisted the nanoparticle to be concentrated into the nucleus through active targeting functionality. Most importantly, the polymeric NPs that are capable of switching their surface charge from negative to positive at an intracellular acidic microenvironment might be considered as a potential candidate for nucleus targeting delivery due to their interactions with anionic forms of proteins. Recently, rational design and composition of some hybrid nanoparticles have been proposed for nuclear targeting and drug delivery.124,125 The advantages of hybrid nanoparticles are high payloads as well as noninvasive imaging for tracking their biological distribution in vivo. Jana et al. reported a hybrid nanoparticle composed of acridin-9-methanol fluorescent organic nanoparticle which is effective for simultaneous nucleus targeting, light induced controlled drug release, and optical imaging of a cell.126 The hydrophobic drug chlorambucil was physically loaded into the core of the acridin-9-methanol fluorescent nanoparticle. Upon laser (500 nm) irradiation of the outer shell, the nanoparticle starts degrading, thereby potentially releasing the entrapped therapeutics in the nucleus (Figure 9). This system, having dual effects, generated from therapeutic drug and laser exhibited higher therapeutic efficacy than that of free drug studied in HeLa cells in vitro. However, their system has limitations for in vivo applications due to lower absorption wavelength. The incorporation of therapeutic molecules along with peptides shows the best therapeutic efficacy and less toxicity in nuclear targeting drug delivery systems.127 Over the last decades many peptides 31 ACS Paragon Plus Environment


have been reported which show great potential in targeting nucleus and penetrating the nuclear membrane.128,129,130,131,132,133

Figure 9. A schematic presentation of photocontrolled nuclear-targeted delivery of chlorambucil by photo-responsive organic nanoparticles of acridin-9-methanol. The therapeutic agent acridin9-methanol starts to release from the nanoparticle once light source are applied to the cell. Reproduced with permission from ref 126. Copyright 2013 American Chemical Society.

6. Conclusions and perspectives This review aims to cover the most recent findings and advancements on approaches, methodologies and strategies for the targeted delivery of therapeutics to subcellular organelles. Considering the progress and advancement on drug delivery systems that focused on development of tools, materials, methods and technology, it is now, more than ever highly important to pay attention to the development of more sophisticated strategies that will facilitate the control of the sub-cellular localization of therapeutics. There is no doubt that specific therapeutics show


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advantageous activity in a specific subcellular site, therefore the particular therapeutic is required to target to the specific site of a targeted cell. It is also well understood that there are various factors such as size and surface charge of a particle, molecular weight and binding affinity of the therapeutics etc. play vital roles to drive the therapeutics to a specific targeted site. The rational design of a carrier or vehicle plays a critical role in every stage such as vascular circulation, organ deposition, extracellular targeting and intracellular localization. Selection of an appropriate material is also very important since various factors such as systemic circulation, retention time, stability during circulation, interaction with biomolecules, and localization in the cells are closely related with the physical and chemical properties of the designed formulations. The major objectives of designing such formulations are; enhanced biocompatibility, reduced toxicity, regulated nonspecific distribution/accumulation and higher localization by the targeted organ or tissue. For example, the retention time of a therapeutic is significantly enhanced when it is incorporated with a polyethylene glycol or its derivative. Therefore, the small molecules having significantly low half-life and circulation time have been widely studied in order to overcome the limitations through polymeric modification. The inorganic nanocrystals such as quantum dots are also potential for enhancing retention time as reported by many investigators, and more importantly they can be considered as real-time imaging agents for observing biodistribution and organ accumulation noninvasively. Several polymers as well as light induced photothermal agents have been demonstrated to be very effective for overcoming the resistance in the extracellular matrix. Nanomaterials such as graphene derivatives and gold nanoparticles have also shown potentials for generating adequate heat to facilitate their penetration into the cells through overcoming the barrier of the extracellular matrix with significantly lower toxicity. For targeting the cell membrane of a particular diseased cell, an appropriate targeting ligand should be chosen



to decorate the surface of the nanoparticles that could directly bind to the receptor of a targeted cell. Several types of peptides have been reported in literature which are significant for distinguishing cancer and normal cells, and even subtype of cells. Designing a formulation with the right targeting ligand can limit random and non-specific distribution of therapeutics thus enhances their accumulation and concentration into a targeted cell. Once the therapeutic is uptaken by a cell, barriers in the cytoplasm could prevent it from traveling to mitochondria or nucleus. The smart polymeric materials for example, poly-l-lysine, has been found to be an effective weapon for endosomal escape could be used. In a recent review, Lu et al. explained the structure of mitochondria, its functions and roles in cancer, as well as the strategies for delivering drugs and macromolecules to mitochondria.134 Here, in this review we have discussed many potential and promising therapeutic molecules that have been used for targeting respective intracellular organelles such as TPP for mitochondria targeting and dexamethasone for nucleus targeting. This review could provide a broad scenario on progress on targeting therapeutics delivery, limitations of current approaches and possible strategies to overcome these limitations. The authors hope that the insights of this review will provide a useful stimulus to encourage future research and development of next-generation intra- or subcellular-targeted therapeutics delivery systems. In conclusion, development of sub-cellular targeting strategies such as nucleus targeting delivery systems will advance the progress in gene delivery research, and mitochondrial targeting delivery systems will be widely useful in treating various diseases included but not limited to age, cardiomyopathy, diabetes and cancer. Acknowledgements This work was supported in part by NIH Grants R01HL122238 (JRM), R01HL133153 (JRM), R01HL102368 (JRM), and The Leading Human Resource Training Program of Regional Neo 34 ACS Paragon Plus Environment

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industry through the National Research Foundation of Korea (NRF) funded by the Ministry of Science,

ICT

and

future

Planning

(NRF-2016H1D5A1910188

AND

NRF-

2018R1D1A1A09083269) to YK Lee, and Industrial Technology Innovation Program [10060, Externally Actuatable Nanorobot System for Precise Targeting and Controlled Releasing of Drugs] funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea).

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Biomaterials and Bioengineering Approaches for Mitochondria and Nuclear Targeting Drug Delivery Md Nurunnabi, Zehedina Khatun, Abu Zayed Md Badruddoza, Jason R. McCarthy, Yong-kyu Lee, Kang Moo Huh


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Biomaterials and Bioengineering Approaches for Mitochondria and

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