Enabling Nanohybrid Drug Discovery through the Soft Chemistry

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Review

Enabling Nanohybrid Drug Discovery through the Soft Chemistry Telescope Goeun Choi, Huiyan Piao, Myung Hun Kim, and Jin-Ho Choy Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b02971 • Publication Date (Web): 11 Oct 2016 Downloaded from http://pubs.acs.org on October 15, 2016

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Industrial & Engineering Chemistry Research

Enabling Nanohybrid Drug Discovery through the Soft Chemistry Telescope Goeun Choi, Huiyan Piao, Myung Hun Kim, Jin-Ho Choy* Center for Intelligent Nano-Bio Materials (CINBM), Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, Korea

KEYWORDS Layered inorganic compounds, Layered double hydroxides, Anionic Clays, Nanoconvergence, Nanomedicine, Drug and gene delivery systems, Chemotherapy.

ABSTRACT

An attempt is made to describe an emerging convergence science, “Nanomedicine”. In particular, inorganic compounds like anionic clays, LDHs (layered double hydroxides), with nanoscale are underlined how they could interact with bioactive and/or drug molecules to form novel intercalative hybrid drug systems with biocompatibility, imaging and targeting functions eventually for gene and/or drug delivery. In this regard, LDHs are focused as an important inorganic biomaterial for drug and gene delivery carriers with very high additive value in the

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near future especially in biomedical industries including pharmaceutical, cosmeceutical, and nutraceutical ones compared to any other industrial applications. In this review, the drug delivery systems based on clay nanoparticles are described in terms of nano-toxicity, intercellular uptake mechanism and intracellular trafficking pathways in in-vitro, and finally passive and active targeting functions in in-vivo. And several studies highlighting recent advances of chemo- and gene-therapies with nano LDHs are also discussed in the view point of state-of-the-art convergence technology based on nanomedicine.

1. Introduction According to the MIT technology review in 2006,1 there were two important research fields of NT-BT convergence, “Nanomedicine” and “Nanobiomechanics”, out of ten emerging technologies, those which have been most intensively studied so far. Since nanobiomechanics is out of the scope of this review, nanomedicine is only described as one of the emerging nano-bioconvergence fields. Nanomedicine comes to be considered as a medicine combined with nanoscience and technology. As described in the document of European Technology Platform, however, “Nanomedicine” is defined as the application of nanotechnology to health. It takes advantage of newly induced physico-chemical and biological properties of materials at the nanoscale.2 As shown in Figure 1, nanomedicine is composed of various research fields such as drug delivery, drug and therapy, in-vivo imaging, in-vitro diagnostics, active implants and biomaterials.3 However, drug delivery is thought to be the most important field in nanomedicine in the viewpoint of the paper numbers published in academic journals, and the number of patents applied and filed in the world.1 The most important of all in research is the designing of drug delivery systems depending upon the research goal desired such as controlled release,4-6


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enhanced solubility,7,8 improved bioavailability,9,10 taste masking,11 overcoming drug resistance,12 passive targeting,13-15 and active targeting.16,17 More recently, many efforts have been made to explore hybrid drug delivery devices functionalized with passive- and active targeting moieties such as PEG and antibodies for in-vivo experiments.18-21 In order to realize such drug delivery devices with desired functions, it is most essential to develop a biocompatible drug delivery carrier (Figure 2). Among various nanovehicles, the most widely and intensively studied one is thought to be the liposome, which is nothing but a self-assembly of amphiphiles. And drug or gene molecules are encapsulated in the core of liposome, whereas its external surface can be modified by conjugating with PEG, antibodies, and dyes depending upon one’s research goals and requirement. In addition to this, there are several other drug delivery carriers studied like polymeric nanoparticles and micelles, dendrimers, and even inorganic compounds such as silica nanoparticles, titanium oxides and iron oxides with various sizes and morphologies, carbon nanotubes, nanohorns, and graphenes, etc.22-25 But in this review, we focus only on anionic clays with lamellar structure, namely, the layered double hydroxides (LDHs), which have been frequently utilized as precursors for inorganic synthesis,26,27 catalyst and catalyst-supports,28,29 sensors,30,31 gas-separation membranes,32,33 and excipients for drug formulation and even medicines for gastric hyperacidity and dyspepsia.8,34,35 Recently, LDHs have attracted a great attention as a nanovector thanks to its superior biocompatibility, controllable drug loading capacity, enhanced cellular permeability, and unusual drug resistant property.12,36-40 The general chemical formula of LDH can be expressed as [M2+1-xM3+x(OH)2]x+(An)x/n⋅mH2O (where M2+: divalent metals such as Ca2+, Mg2+, Zn2+, and Ni2+, etc., M(III): trivalent ones such as Al3+, and Fe3+, etc., An-: anionic species such as CO32-, NO3-, Cl-, and SO42-, etc.,


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0 OH- > F- > Cl- > Br- > NO3- > I-.42 Based on the chemical routes above, various biologically active molecules, such as nucleotides,36,37,43,44 anticancer drugs,20,45-47 antiinflammatory drugs,48 antibiotics,49 and vitamins50,51 have been intercalated into LDHs to develop novel Bio-LDH nanohybrids with various functions. Anion (drug) content in LDH can be modified simply by controlling the ratio of M2+ and M3+ in the lattice, since it directly relates to the magnitude of layer charge density and at the same time, that of anion exchange capacity. More recently, the nano-LDH with an average size of ~100 nm was found to be biocompatible and targetable to the tumor tissues and cells, due to its particle-size dependent


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targeting function, which could be ascribed to its enhanced permeability and retention (EPR) effect and clathrin-mediated endocytosis mechanism.34,52,53 As previously demonstrated by Choy’s group, not only the LDH nanovehicle but also the drug(MTX)-LDH and gene(siRNA)LDH nanohybrids did not induce any liver toxicity and morphological abnormalities due to liver injury, which was confirmed by examining the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the plasma serum, and by performing the histopathologic analysis of hematoxylin-eosin (H&E) stained liver sections, respectively.19,47 As shown in Figure 4, soft chemical routes to Bio-LDH nanohybrid materials, such as co-precipitation, ion-exchange, calcination-reconstruction, and exfoliation-reassembling are summarized.54 Especially, in Figure 4(A), co-precipitation is the most efficient and convenient route to Bio-LDH nanohybrids. In addition, ion-exchange reaction, as shown in Figure 4(B), is also widely used for preparing Bio-LDH nanohybrids via replacing interlayer anions, such as NO3- or Cl- ions with desired drug- and bio-molecular- anions. Though calcinationreconstruction and exfoliation-reassembling routes (Figure 4(C) and (D)) can be an alternative strategy, but not that desirable in terms of phase purity.54 Based on these synthetic strategies, one can prepare a hundreds of different kind of Bio-LDH nanohybrids applicable for drug and gene delivery systems.55-59 Various examples highlighting recent advances of chemo- and genetherapy are demonstrated in the viewpoint of nanomedicine, along with the emerging issues related to the selective and stimuli-responsive delivery of Bio-LDH hybrids in in-vitro and invivo.


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2. Endocytic and exocytic pathways of nanoparticles Endocytosis is a kind of process whereby alien molecules or nanoparticles are wrapped with membrane protein and permeated inside the cell in the form of vesicles or vacuoles. There are, however, multiple endocytic pathways in mammalian cells, such as macropinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis, lipid rafts-mediated endocytosis and clathrin- & caveolae-independent endocytosis one. According to the studies on cellular uptake behavior of LDH nanoparticles, it becomes clear that the endocytic pathways are dependent on the particle size. Therefore, the size controlling of LDH nanoparticles is essential in the designing of drug and gene delivery systems.

2.1. Intercellular uptake mechanism of LDH nanoparticles The most attractive feature is that LDH nanoparticles smaller than ~250 nm can be internalized inside the cells through clathrin-mediated endocytosis,52,53 which is the most common endocytic behavior in all mammalian cells, and eventually play a role as a drug delivery carrier with targeting function due to the specific endocytic pathway. This is in good agreement with such a low cellular uptake of LDH nanoparticles of 300~350 nm in size, which indicates a non-selective permeation pathway of large particles. According to the immunofluorescence microscopy and confocal laser scanning microscopy studies on osteosarcoma cells (MNNG/HOS) treated with FITC-LDH (~100 nm), it was experimentally well demonstrated that LDH nanoparticles were permeabilized into cells via clathrin-mediated endocytosis (Figure 5(A)). MNNG/HOS cells treated with FITC-LDH, and clathrin antibody and its secondary antibody conjugated with dye showed that FITC-LDHs were mainly present in the cytosol, and


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highly colocalized with the clathrin protein (Figure 5(B)). This result clearly suggests that the cellular internalization mechanism of LDH nanoparticles is nothing to do with the caveolaemediated endocytosis, but mainly related to the clathrin-mediated one.52 One more thing to underline here is that the LDH delivery carrier can be considered as the promising alternative to provide a way of overcoming the drug resistance in chemotherapy, thanks to the clathrin-mediated endocytosis which will be discussed in section 2.2. (Figure 6(A)).12

2.2. Intercellular uptake mechanism of MTX-LDH hybrid nanoparticles: Clathrinmediated endocytic route to bypass the drug resistance It has been already well understood that methotrexate (MTX) can be internalized inside the cells via the reduced folate cycle (RFC) and the folate receptor (FR) sites, but its uptake rate is so low that a high dosage is required for chemotherapy.60 The challenging issues in MTX chemotherapy are not only the low uptake rate, very short plasma half-life, but also the drug resistance when repeatedly administered.60 MTX has been prescribed, as an anticancer drug, for choriocarcinoma, including osteosarcoma, acute leukemias, breast cancer, cervical cancer, and even for rheumatoid arthritis. When MTX is once uptaken by the cell through the specific pathways, RFC and FR, it inhibits the activity of dihydrofolate reductase (DHFR) in the cell, since it acts as a folate antagonist. If the cytosolic enzyme (DHFR) becomes conjugated with MTX and eventually deactivated, the enzymetic reduction of dihydrofolate to tetrahydrofolate in folate cycle, which is coupled with DNA synthesis and cell proliferation, is stopped and eventually blocks the folate cycle.46 And as


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a consequence, MTX inhibits the production of thymidine, de novo DNA synthesis, and cell division, and drives the cell to death (Figure 6(B)).57,60-63 Though its anticancer mechanism is clear as described above, an important drawback of MTX is thought to be the high administration dose required for an effective chemotherapy due to its low cellular uptake rate, very short plasma half-life and high ratio of efflux rate/influx rate. According to Gottesman’s reports, the efflux of MTX could be increased via ATP-binding cassette (ABC) subfamily transporters such as ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, and ABCG2 due to the upregulation of drug-efflux proteins.62,64 To overcome these limitations, attempts have been made to intercalate MTX into LDH layers to prepare new MTX-LDH hybrid drug.12,20,47 As shown in Figure 6(C), the effect of intact MTX drug or MTX-LDH hybrid drug on the inhibition of cancer cell proliferation was evaluated in both cell culture lines, MTX sensitive one (wild-type HOS cells) and MTX resistant one (HOS/Mtx cells), respectively, on the basis of MTT assay. When intact MTX was treated, the cell proliferation was remarkably inhibited in wild-type HOS cells compared to HOS/Mtx cells, suggesting that the drug resistance led to a reduction in drug efficacy. Whereas the cell viability of MTX-LDH was strongly decreased with the same degree upon variation of drug concentrations, whatever the cells were drug-sensitive or drug-resistant. This result indicates that LDH carrier can smuggle MTX drug molecules not through the typical MTX permeation routes (RFC and FR) but through the chlathrin-mediated endocytic pathway. It is, therefore, expected that the MTX-LDH hybrid can be a potential anticancer drug to overcome drug resistant problems faced on chemotherapy. The drug resistant mechanism has been systematically investigated to understand how the LDH carrier could bypass the cellular uptake pathway of MTX, RFC and FR sites, and deliver drug inside the cells without experiencing drug resistance and eventually enhance the anticancer activity.12


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2.3. Intracellular trafficking pathways of LDH nanoparticles After unveiling the intercellular uptake mechanism of LDH nanoparticles, an attempt was again made to understand their fate when they were once internalized in the cell. Choy’s group has systematically studied the intracellular trafficking pathways of LDH nanoparticles with two different-sizes (50 nm and 100 nm) conjugated with FITC in HOS cells (Figure 7(A), (C)).13 And the trace of intracellular trafficking behavior was investigated on the basis of immunofluorescent staining method. To do this, the cells were incubated with primary antibodies against early endosomal antigen-1 (EEA-1), RAS related protein (Rab7), lysosomal-associated membrane protein-1 (LAMP-1), trans-Golgi network membrane protein (TGN38), and integral protein of the ER (calnexin) as the marker of early endosomes, late endosomes, lysosomes, Golgi complex, and Endoplasmic Reticulum, respectively. To quantify colocalization between LDH nanoparticles and individual antibodies, the percentage of colocalized images was then calculated from the amount of overlaped areas between total green area and red one using Crop option. As shown in Figure 7(B), the LDH particles with an average size of 50 nm were found almost equally in the lysosomes and the Golgi apparatus in the time range of 0.5-6 h, suggesting their typical endocytic pathway of “early endosome-late endosome-lysosome”, rather than an exocytic pathway corresponding to “early endosome-Golgi-ER-Golgi”. As shown in Figure 7(D), however, the 100 nm LDH particles were primarily localized in the early endosomes at 0.5 h, and after 1-24 h incubation period, obviously high amount was observed in the Golgi complex compared to the lysosomes. This is a clear evidence that the large particles (100 nm) seem to be directly discharged by the exocytosis pathway of “early endosome-Golgi-ER-Golgi”, rather escaping from typical endosomal and lysosomal degradations. It is, therefore, highly expected


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that the present delivery carrier (LDH) would be very advantageous for gene delivery system, due to its unique size-dependent distinction of intracellular trafficking pathway with endosomal and lysosomal escapes.

2.4. Intracellular trafficking pathways of gene-LDH hybrid nanoparticles: Size-dependent exocytic pathway to escape from endosomal and lysosomal degradation It is interesting to check in this step whether the size-dependent exocytic behavior of LDH carrier is really efficient in gene delivery. And therefore, the Survivin siRNA (siSurvivin) was intercalated into KB cells to prepare a gene delivery system. The siSurvivin (Bioneer Co.) selected

is

composed

of

19

nucleotides

with

the

sense

strand

(5’-

AAGGAGAUCAACAUUUUCA-3’) and the antisense strand with a complementary sequence (5’-UGAAAAUGUUGAUCUCCUU-3’), which is a gene to target the proto-oncogene Survivin, a kind of inhibitor of apoptosis (IAP) gene family inducing cancer specific cell death.19,65 To confirm whether post-translational regulation of Survivin could influence on the cancer cell proliferations, the siSurvivin-LDH nanohybrid was transfected into the KB cells. And the cell survival was examined on the basis of MTT assay as shown in Figure 8. As can be seen clearly that the cell proliferation was strongly reduced by ~53% when the siSurvivin-LDH nanohybrid was incubated in the KB cells. On the other hand, negligible change in cell survival could be observed for the siSurvivin only treated.

3. Active- & Passive Targeting In order to improve the therapeutic efficacy, drug molecules must be target-delivered to


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malignant tissues or cells with passive and active targeting strategy. As already described, the LDH nanoparticles with an average size of 100 nm can be considered as the potential drug delivery carrier with targeting functions to overcome drug resistance, since the cellular uptake rate is so high comparable to other carriers thanks to the clathrin-mediated endocytosis. Though it has been already well known that the LDH nanoparticles are preferentially internalized through the cellular membrane, an active targeting strategy was made to by conjugating the tumor targeting ligand such as folic acid on the surface of LDH nanoparticles.66 As shown in Figure 9, the cellular uptake of MTX in FA-LDH nanohybrid was strongly enhanced compared to that in LDH which resulted in an increased anticancer effect, as well demonstrated in KB cells. According to the literature study,20,46,47 the enhanced permeability and retention (EPR) effect, due to the voids in vasculature in tumor tissues, is mainly responsible for the passive targeting. To confirm EPR effect, the drug selectivity to tumor tissues can be determined by blood circulation and extravasation after the administration of nanohybrid drugs through oral or injection route. To realize such a passive targeting strategy, the drug of interest must be designed and hybridized with drug delivery carrier to have a long circulation half-life for sufficient delivery by controlling the parameters, such as particle size, surface charge, colloidal stability, and other biophysical properties of delivery carrier.45-47,67-69 In this regard, Choy’s group has intensively studied on the passive targeting effect of the size-controlled anticancer drug-LDH, and found that the hybrid drug of MTX-LDH with ~100nm could strongly inhibit the tumor growth compared to the controls.20,47 As shown in Figure 10, the pristine LDH and MTX-LDH nanoparticles were suspended in decarbonated water with a size of 100 nm.20,47 When each of them was added to the Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine


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serum (FBS), a colloidal suspension could be realized with an average particle size of 90 and 100 nm, respectively.20 As represented in Table S1, LDH nanoparticles (100–200 nm) tend to be accumulated more in tumor cells or tumor tissues than in normal cells or normal tissues, indicating an enhanced tumor targeting efficiency surely due to the EPR effect.52,57,70-74

4. Chemo- & Gene- therapy 4.1. Chemotherapy Cancer is a mondial health problem, affecting every region and socioeconomic level. Moreover, cancer proves fatal in many cases and today, it comes up to approximately 1 in every 7 deaths in the world – more than any other serious contagious diseases like AIDS, tuberculosis, and etc. As well documented,75 there were around 8.2 million cancer deaths in the world out of 14.1 million cancer patients estimated in 2012. Moreover, the welfare benefits in each nation will become unendurable to keep up such a cancer burden continuously, because this financial burden will be added to the fiscal budget after all. According to the forecast by the American Cancer Society,75 new cancer cases of about 21.7 million and cancer deaths of 13.0 million will occur only in the year of 2030, simply due to the increase of the elderly population.75 In this respect, the drug delivery research can be considered as really an important area, comparable to the drug discovery, in the field of nanomedicine, and this is the reason why drug delivery research is as active as ever and still an important issue in biomaterials and drug delivery communities.76 As shown in Table S1, there have been researches to develop new nanoscale DDS for chemotherapy with targeting functions of LDHs nanoparticles. As already demonstrated in the


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previous reports by Choy’s group,12,20,46,47 they were very successful in encapsulating the anticancer drug, methotrexate, into layered double hydroxide (LDH) to form methotrexate-LDH (MTX-LDH) nanohybrid, whose particle size was determined to be 127±25 nm. According to the trypan blue assay, the cell viability in culture was more strongly reduced upon incubating with MTX-LDH than with MTX only. The IC50 value of MTX in the tumorogenic osteosarcoma MNNG/HOS cells was about 2.5 times higher than that of MTX-LDH only indicating that the MTX-LDH nanohybrid particles could come across the cell membrane more effectively than MTX only, resulting in an improved efficacy. Therefore, the drug efficacy of MTX-LDH was turned out to be much better than that of MTX only, due to an enhanced cellular uptake behavior of the MTX-LDH nanoparticles. Furthermore, LDH nanoparticles themselves do not influence the cell viability up to such a high concentration of 500 µg/mL (Figure 11).52 Also, the drug efficacy of MTX-LDH was evaluated according to the cell viability test (MTT assay) in the human breast adenocarcinoma cell culture line (MCF-7). The IC50 value of MTX-LDH was also considerably lower than that of MTX only, indicating that the drug efficacy of MTX-LDH was much more enhanced than MTX only as the same as in the previous example of MNNG/HOS cell line.70 In addition, Oh et al. also reported that MTX-LDH could have higher drug efficacy than MTX only even under the condition of a reduced dose and a shorter incubation time based on the results of MTT assay by evaluating the cell viability in the human osteosarcoma cells (Saos-2 and MG-63).57 In addition to the MTX-LDH hybrid drug, Choy’s group developed an another new nanohybrid system by intercalating 5-fluorouracil (5-Fu) into LDHs, and its IC50 value of 5-FuLDH with an average particle size ~80 nm was also about 2.5~4.3 fold lower than that of free 5Fu depending on the cell lines such as human lung adenocarcinoma cells (A549), human liver


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carcinoma cells (Hep 1), and human osteosarcoma cells (HOS), indicating that the drug efficacy was strongly enhanced due to the well-defined endocytic mechanism of LDH nanocarrier.71 A simple co-precipitation route to new LDH hybrid of zinc(II) phthalocyanine tetra-asubstituted with 4-sulfonatophenoxy groups (ZnPcPS4) was also demonstrated by Huang, et al.73 The IC50 value of ZnPcPS4-LDH toward human hepatocarcinoma cells (HepG-2) was determined to be more than 24-folds lower than that of ZnPcPS4 indicating that the photocytotoxicity of ZnPcPS4 in the ZnPcPS4-LDH nanohybrid toward HepG-2 cells was significantly enhanced. This is surely due to the fact that the negatively charged ZnPcPS4 itself may not be favorably uptaken by the cells, since the cellular membrane is also negatively charged resulting in repulsive interaction between them. On the other hand, the ZnPcPS4-LDH nanohybrid particles were permeated inside the cells more efficiently, because of the effect of charge neutralization upon hybridization between ZnPcPS4 and LDH. In addition, it was found that ZnPcPS4 molecules could be efficiently released out from the LDH lattice, and be highly photosensitizing after endocytosis. As a similar example of anticancer drug-LDH nanohybrid, the doxorubicin (DOX)-loaded LDH magnetic nano-hybrids (MNHs) were demonstrated. It was also found that the MNHs formulations with an average particle size ~240 nm could enhance the therapeutic performance for thermo-chemotherapy in the human cervical cancer cells (Hela).74 Most recently, in-vivo studies have been made for anticancer efficacy of the MTX-LDH nanohybrid in the orthotopic tumor models of breast cancer and cervical one, respectively.20,47 And for the first time, the LDH carrier was prepared in a colloidal form and used for injectable nanomedicine in orthotopic model,47 which is thought to be clinically more pertinent and therefore more predictive to estimate drug efficacy and/or toxicity than the standard xenograft models.77 The antitumor activities, biodistributions, in-vivo toxicity were systemically evaluated


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after intraperitoneal (i.p.) injection of MTX-LDH into each orthotopic mice model (Figure 12). The MTX-LDH nanohybrid system exhibited remarkably high antitumor efficacy in both in-vivo models due to the EPR effect. As shown in Figure 12A(a) and B(a), the therapeutic efficacy of MTX-LDH compared to pure MTX was 74.3% and 66.4% reductions in tumor volume after drug administration in the orthotopic breast cancer and the orthotopic cervical cancer models, respectively. Interestingly, the tumor to liver ratio of MTX-LDH was found to be 6-fold higher in the former model and 3.5-fold higher in the latter one, respectively, than that of pure MTX after intraperitoneal injections. (Figure 12A(b) and B(b)). Considered the tumor-to-liver ratio is an essential indicator in terms of therapeutic efficacy and safety profile, the enhancement of this ratio for MTX-LDH indicates a clear sign of its high potential as a safe and effective systemic delivery system for chemo-therapy. Furthermore, the animal groups treated with MTX-LDH showed a 100% survival rate, suggesting that MTX-LDH obviously inhibited the growth of tumor volume in the orthotopic breast cancer model and eventually increased the survival rate compared to free MTX (Figure 12A(c)). In orthotopic cervical cancer model, the tumor growth was also significantly inhibited by the administration of MTX-LDH compared to the control. And moreover, no in-vivo toxicity could be observed by monitoring the body weight of the mice with respect to time (Figure 12B(c)). Wei & Yan’s group have demonstrated the effect of photodynamic therapy (PDT) on the anticancer performance of zinc phthalocyanines-layered double hydroxide (ZnPc-LDH) in HepG2 tumor bearing mice. As shown in Figure 13(A), the ZnPc(1.5%)-LDH hybrid photosensitizer was found to be most superior in terms of in-vivo PDT, i.e., the growth rate of tumor tissue was surpressed to a great extent.55 Wu & Wang’s group were also able to demonstrate an enhanced anticancer efficacy of


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layered double hydroxide-etoposide (VP16-LDH) hybrid on non-small cell lung cancer (NSCLC) xenograft mice model. As shown in Figure 13(B), A549 xenograft tumor bearing mice model was made to study anticancer effect of VP16-LDH and pure VP16. When VP16 only injected, the tumor volume after 21 days was about 2.3 fold bigger than that of the first day, but the tumor volume was almost remained unchanged upon administration of VP16-LDH due to the targeting effect of LDH.56 As mentioned above, the 100 nm sized LDH is proven to be biocompatible and could deliver anticancer agents to the tumor with high selectivity, owing to its specific targeting function thanks to the EPR effect and cellular uptake mechanism of clathrin-mediated endocytosis.52,53,66

4.2. Genetherapy Choy et al. applied the LDH nanoparticles as a non-viral gene delivery vector, for the first time, which could deliver helpful DNA, such as antisense myc (As-myc), into cells. According to their laboratory experiments, negatively charged As-myc with sequence of 5’-d(AACGTTGAGGGGCAT)-3’ gene molecules were shielded by positively charged LDH layers due to those opposite forces, helping them permeate through the cell membrane, since the negatively charged molecules are, in general, repelled by many cells with their similarly charged cell membranes. Choy’s group also showcased LDH carriers in well-designed in-vitro experiments with As-myc-LDH nanohybrid, in which LDHs can smuggle fluorescent molecules into cells and protect intercalated DNA from DNase degradation,37 and intercalate a DNA sequence designed to impede a specific gene of leukaemia cells grown in the laboratory.


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According to the MTT assay, the time-dependent inhibition of proliferation could be observed when the human promyelocytic leukemia cells (HL-60) was incubated with As-myc-LDH, resulting in 65% suppression of cell growth compared to the control as shown in Figure 14(A). O’Hare’s group reported on the cellular uptake of ~20 nm LDH for gene delivery in mouse motor neuron cells (NSC 34). As shown in Figure 14(B), the in-vitro transfection of pEGFP-N1 with the DNA adsorbed phase of the carbonate LDH (DNA-CO3LDH) with the size of 20 nm nanoparticles in NSC 34 cells was confirmed by the expression of GFP from the green fluorescent cells (Figure 14B(a)). Even brighter fluorescent cells could be observed due to the transfection with the DNA adsorbed on the nitrate LDH (DNA-NO3LDH) as shown in Figure 14B(b), while no expression of GFP could be observed with the naked DNA even for 3 days. It is, therefore, concluded that the DNA-NO3LDH phase exhibits more expression of GFP compared to the DNA-CO3LDH one, and as a consequence, DNA molecules in the former seemed to be better protected from the enzymatic degradation than those in the latter, probably due to the fact that a partial intercalation of DNA into the NO3LDH lattice is possible, since the interlayer nitrate ions are labile and exchangeable with DNA, but the carbonate molecular ions in the DNA-CO3LDH phase are inert and thermodynamically very stable and therefore, remain in the lattice without any ion exchange reaction with DNA and consequently, fragile DNA molecules can only be adsorbed on the external surface of the CO3LDH phase rather leaving DNA unprotected.78 Recently, Xu’s group intended to deliver simultaneously 5-Fu and Allstars Cell Death siRNA (CD-siRNA) with the help of LDH delivery carrier for effective cancer treatment. Such a combination therapy of CD-siRNA and 5-Fu, co-delivered by LDH, resulted in less cell viability, particularly at lowered 5-Fu concentrations (Figure 14(C)). When CD-siRNA-5-Fu-LDH was


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treated with the concentration at 1.2 µg/mL of 5-Fu and 40 nM of siRNA, ~ 70% cell death occurred in MCF-7 cells. On the other hand, only 46% cell death could be observed for the case of 5-Fu-LDH only treated in the same cell culture line with the same concentration. This is an evidence that the combination treatment with CD-siRNA and 5-Fu with LDH nanoparticles significantly could suppresses cancerous cell growth, due to the synergy effect of two different drugs by effective induction of cell death in complementary pathways.59 As shown in Table S2, Lu’s group demonstrated that siRNA molecules could be delivered into human embryonic kidney cells (HEK293T) with about 99% efficiency only when they were immobilized in LDH. Such a high efficiency is even better than that of Lipofectamine®.79 Taviot-Guého & Pitard’s group was able to prepare a few modified LDHs and their DNA hybrids such as DNA-Mg-Al-LDH, DNA-Mg-Fe-LDH, and DNA-Mg-Ga-LDH, and to demonstrate that the DNA molecules could be recovered intentionally out of inorganic LDHs by dissolving them under an acidic pH condition, and that those DNA hybrids were taken up by human cervical cancer cells (HeLa).43 According to the Xu’s study, the plasmid DNA (pDNA) was better condensed within P(DMAEMA)-Grafted LDH (PDs-LDH) hybrids, where P(DMAEMA) stands for poly((2-dimethylamino)ethyl methacrylate) and abbreviated as PDs, and protected from the attacks of nucleases. And eventually this PDs-LDH resulted much higher level of delivering genes in various cell lines including monkey kidney fibroblast cells (COS7) and human hepatocarcinoma cells (HepG-2).44 Most recently, Choy’s group has successfully demonstrated in in-vitro delivery systems for the Survivin siRNA (siSurvivin) hybridized with LDH nanoparticles, siSurvivin-LDH, those which were carefully designed to have a passive targeting function by controlling the particle size around ~100 nm, as already discussed in section 2.4.


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And to induce an active targeting function, the surface of LDH nanoparticles were modified by conjugating with a targeting ligand to cancer overexpressing receptor, folic acid (FA-LDH). Based on their synthetic strategy, they were very successful in demonstrating the active and passive targeting functions of LDH nanohybrid particles in in-vitro and in-vivo by either EPR effect and clathrin-mediated endocytosis or folate receptor-mediated endocytosis. Transfected into KB cells or injected into xenograft mice, it was evidenced that siSurvivin-FALDH could resulted in an excellent effect of gene silencing at mRNA and protein levels in invitro, and eventually achieve a 3.0-fold better inhibition in tumor growth than siSurvivin-LDH in in-vivo (Figure 15(A)). Such an anti-tumor effect could be explained by the fact that siSurvivin molecules could be more targeted (1.2-fold higher) to tumor tissues compared to other organs (Figure 15(B)).19

5. Summary In this review, efforts have been made to demonstrate that layered inorganic materials and their intercalative hybrids can open the way of developing new platform technology of drug delivery systems (DDSs) for chemo- and genetherapy in the future. Among various possible inorganic delivery carriers, the layered double hydroxide can certainly be the most potential candidate for that purpose due to its unique features such as excellent biocompatibility, high anion exchange capacity (2-5 meq/g) and pH-dependent solubility, those which are closely related to the loading content of negatively charged drugs and/or therapeutic genes. According to the previous results, LDH with 2-dimensional framework can encapsulate fairly large amount of anionic bioactive molecules like DNA and anticancer drugs upon intercalation reaction, and also penetrate the cellular membrane across the wall barriers and eventually enhance the cellular


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permeation of intercalated drug and/or gene molecules. Though researchers in drug delivery community are still on the way of actively providing in-vivo evidences, the hybridization of biofunctional or bioactive molecules with LDHs would open an emerging research area for advanced drug delivery systems as well as a new area of nanomedicine in the near future.

6. Outlook Various drug delivery technologies are being currently investigated not only in university laboratories but also in industrial research institutes in the world very competitively. Though their research goals of such DDSs in nanomedicine could be slightly different form each other, but should be beyond satisfaction of academic curiosity or money-making, wherever the researches are made, the scientists and engineers working in nanomedical area must be inspired by knowing that all the findings and understandings are also serving the goal of providing for cancer patients in need. In this regard, the present new drug delivery platform based on 2-dimensional inorganic nanovector (LDHs) can be considered as an important new convergence technology emerged out of nanomedicine, especially applicable for chemo- and gene therapy as follows ; (A) the present LDH is biocompatible, dissolvable in a weakly acidic bio-fluid, not accumulated in body, and therefore, least toxic compared to other carriers. (B) the LDH based drug/gene delivery system can (1) enhance the drug efficacy significantly compared to conventional drugs, (2) improve the patient compliance, (3) provide an enormous change in drug safety due to the passive and active targeting effects of biocompatible delivery carrier, (4) overcome multidrug resistance owing to the clathrin-mediated endocytosis in chemotherapy, and (5) provide a specific pathway to escape


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from endosomal and lysosomal degradation of DNA due to the direct exocytosis represented by early endosome-Golgi-ER-Golgi thanks to the intracellular trafficking pathway of the present nanoparticles with ~100 nm, and etc. (C) the rationally designed LDH carriers and their drug delivery hybrids can be chemically well defined on the basis of synthetic strategy, and their particle sizes can be easily controled in a nano-scale to maximize the passive targeting function based on EPR effect, and moreover, their external surface can also be modified with antibodies and tumor-targeting ligands to enhance the active targeting function and etc. depending upon the research goal desired. And therefore, there would be no doubt that advanced or novel drug delivery systems will be the most important challenge for the healthcare of human-being in the future. What should be done is, therefore, to maximize the collaborative research interaction between academia and pharmaceutical industries to understand from fundamentals to drug formulations, and eventually to get the FDA approval.


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ASSOCIATED CONTENT Supporting Information Table S1: in-vitro and in-vivo studies for chemotherapy; Table S2: in-vitro and in-vivo studies for gene delivery. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (2005-0049412).

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2015, 21, 3310-3317. (74) Komarala, E. P.; Nigam, S.; Aslam, M.; bahadur, D. In-vitro evaluation of layered double hydroxide–Fe3O4 magnetic nanohybrids for thermo-chemotherapy. New J. Chem. 2016, 40, 423433. (75) Cancer Facts & Figures 2016, American Cancer Society, Inc., http://www.cancer.org/ acs/groups/content/@research/documents/document/acspc-047079.pdf/. Accessed on Jun 29, 2016. (76) Wagner, V.; Dullaart, A.; Bock, A. K.; Zweck, A. The emerging nanomedicine landscape. Nat Biotech. 2006, 24, 1211-1217. (77) Killion, J. J.; Radinsky, R.; Fidler, I. J. Orthotopic models are necessary to predict therapy of transplantable tumors in mice. Cancer Metastasis Rev. 1999, 17, 279-284. (78) Li, S.; Li, J.; Wang, C. J.; Wang, Q.; Cader, M. Z.; Lu, J.; Evans, D. G.; Duan, X.; O’Hare, D. Cellular uptake and gene delivery using layered double hydroxide nanoparticles. J. Mater. Chem. B. 2013, 1, 61-68. (79) Ladewig, K.; Niebert, M.; Xu, Z. P.; Gray, P. P.; lu, G. Q. M. Efficient siRNA delivery to mammalian cells using layered double hydroxide nanoparticles. Biomaterials. 2010, 31, 18211829.


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Figure 1. Discovering a star drug delivery system in galaxy of “Nanomedicine” through the hybrid space telescope.


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Figure 2. Various nanovehicles for biomedical applications: (A) layered double hydroxide (LDHs), (B) dendrimer (Figure reprinted with permission from ref 22. Copyright 2005 American Chemical Society.), (C) liposome (Figure reprinted with permission from ref 23. Copyright 2013 American Chemical Society.), (D) polymeric micelles (Figure reprinted with permission from ref 24. Copyright 2011 American Chemical Society.), and (E) iron oxide (Figure reprinted with permission from ref 25. Copyright 2005 American Chemical Society.).


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Figure 3. Schematic crystal structure of layered double hydroxides (LDHs) (A) top view parallel to ab- plane and (B) side view perpendicular to c- axis.


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Figure 4. Soft chemical routes to lattice engineered 2D nanohybrids. (A) Ion-exchange, (B) coprecipitation, (C) calcination-reconstruction, and (D) exfoliation-reassembling.


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Figure 5. Intercellular uptake mechanism of the LDH nanoparticles: (A) schematic illustration of the clathrin-mediated endocytosis, and (B) Confocal microscopy: colocalization of FITC-LDH and clathrin in MNNG/HOS cells. Localization of (a) nucleus, (b) clathrin, and (c) FITC-LDH, the merged image (d) in MNNG/HOS cells. Cells were incubated with FITC-LDH for 2 h, treated with clathrin antibodies, and stained by TR and DAPI. Scale bar represents 10 µm. (Figure reprinted with permission from ref 52. Copyright 2006 American Chemical Society.).


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Figure 6. (A) The mechanism of action of MTX-LDH nanohybrid for bypassing drug resistance (Figure reprinted with permission from ref 12. Copyright 2010 Royal Society of Chemistry.), (B) the anticancer mechanism of methotrexate (MTX) (Figure reprinted with permission from ref 72. Copyright 2012 John Wiley & Sons, Inc.), and (C) Comparative growth inhibition profile of LDH, free MTX, and the MTX-LDH nanohybrid in wild-type HOS and HOS/Mtx cell lines (Figure reprinted with permission from ref 12. Copyright 2010 Royal Society of Chemistry.).


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Figure 7. (A) Schematic diagram of intracellular trafficking pathway of LDH nanoparticles with 50 nm size in HOS cells, (B) quantitative colocalization analysis of FITC-LDH of 50 nm with the five intracellular compartments, (C) schematic diagram of intracellular trafficking pathway of LDH nanoparticles with 100 nm size in HOS cells, and (D) quantitative colocalization analysis of FITC-LDH of 100 nm with the five intracellular compartments (Figure reprinted with permission from ref 13. Copyright 2012 Elsevier.).


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Figure 8. In-vitro comparison of Survivin inhibition efficacy of (A) siSurvivin and (B) siSurvivin-LDH in KB cells by MTT assay (Figure reprinted with permission from ref 19. Copyright 2016 John Wiley & Sons, Inc.).


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Figure 9. Schematic illustration of FA grafting reactions; (A) APS coupling and (B) FA conjugation reactions. And (C) cell proliferation/viability of MTX-LDH and MTX-FA-LDH treated KB and A549 cell lines (Figure reprinted with permission from ref 66. Copyright 2009 John Wiley & Sons, Inc.).


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Figure 10. The particle size distribution and the high-resolution transmission electron microscopy images of (A and B) pristine LDH and (C and D) MTX-LDH in DMEM with 10% FBS, respectively (Figure reprinted with permission from ref 20. Copyright 2016 Dove Press Ltd.).


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Figure 11. Cell viability/cytotoxicity of MNNG/HOS cells treated with LDH (▲), MTX (○), and MTX-LDH (●), as monitored by trypan blue exclusion, with respect to drug concentration (Figure reprinted with permission from ref 52. Copyright 2006 American Chemical Society.).


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Figure 12. (A) Orthotopic breast cancer mice model by i.p. injection (Figure reprinted with permission from ref 47. Copyright 2014 Nature Publishing Group.): (a) antitumor activity, (b) biodistribution studies for tumor-to-liver ratio of MTX, and (c) survival rates of tumor-bearing mice treated as in (a), and (B) Orthotopic cervical cancer mice model by i.p. injection (Figure reprinted with permission from ref 20. Copyright 2016 Dove Press Ltd.): (a) antitumor activity, (b) biodistribution studies, and (c) body weight changes of tumor-bearing mice treated as in (a).


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Figure 13. (A) The tumor growth curves of the six groups of mice after treatment. The tumor volume was normalized to the initial size; the error bar was based on standard deviation of mice per group (Figure reprinted with permission from ref 55. Copyright 2014 John Wiley & Sons, Inc.), and (B) The in-vivo efficacy of VP16 and VP16-LDH on mice bearing A549 xenografts. Tumor growth curves of mice treated with PBS, VP16 and VP16-LDH (*p < 0.05, **p < 0.01 compared to control group) (Figure reprinted with permission from ref 56. Copyright 2016 Elsevier.).


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Figure 14. (A) Effect of As-myc and As-myc-LDH nanohybrid on the growth of HL-60 cells (Figure reprinted with permission from ref 37. Copyright 2000 John Wiley & Sons, Inc.), (B) fluorescence microscopic images of cells with pEGFP-N1 expressed 60 h after transfection using DNA modified (a) 100 µg/ml CO3LDH, and (b) 100 µg/ml NO3LDH (Figure reprinted with permission from ref 78. Copyright 2013 Royal Society of Chemistry.), and (C) schematic diagram of the LDH co-delivery system to co-load 5-Fu and siRNA, and MTT assay analysis of effects of treatments with 5-Fu, 5-Fu(10)-LDH, CD-siRNA-LDH, and CD-siRNA-5-Fu-LDH on the viability of MCF-7 cells at the 5-Fu concentration from 0 to 9.6 µg/mL and the CD-siRNA concentration at 40 nM in all relevant treatments for 72 h at 37 °C. Data represent mean ± SD (n = 5). **indicated p < 0.05 versus 5-Fu treatment, *p < 0.01 versus 5-Fu treatment. ****p < 0.0001 versus 5-Fu treatment (Figure reprinted with permission from ref 59. Copyright 2014 Elsevier.).


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Figure 15. (A) In-vivo anti-tumor effect : tumor growth inhibition (n=6 per group). Tumor volumes were measured at 2-days interval. Tumor sizes are presented as means±SE. *p

Enabling Nanohybrid Drug Discovery through the Soft Chemistry

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