Design of a Novel Theranostic Nanomedicine (III): Synthesis and Physicochemical Properties of Tumor-Targeting Cisplatin Conjugated to a Hydrophilic Polyphosphazene.

PURPOSE: A new theranostic nanomedicine involving anticancer-active cisplatin moiety was designed to study its tumor-targeting properties as well as its drug efficacy and toxicity. METHODS: A cisplatin carrier polymer was prepared by grafting equimolar polyethylene glycol of a molecular weight of 550 (PEG550) and aminoethanol to the poly(dichlorophosphazene) backbone. Cisplatin was conjugated to the carrier polymer using cis-aconitic acid as a linker. RESULTS: The cisplatin-loaded polyphosphazene, named "Polycisplatin" was found to be amphiphilic in aqueous solution and self-assembled into nanoparticles with an average particle size of 18.6 nm in diameter. The time-dependent organ distribution study of Cy5.5-labeled Polycisplatin in the A549-tumor-bearing mice exhibited a high tumor selectivity of Polycisplatin by EPR effect despite the relatively small particle size. In order to compare the in vivo efficacy of Polycisplatin and cisplatin, their xenograft trials were performed using nude mice against the human gastric cell line MKN-28. Polycisplatin exhibited slightly less tumor suppression effect compared with cisplatin at the same dose of 1.95 mg Pt/kg, which is the maximum tolerate dose of cisplatin, but at the higher double dose of 3.9 mg Pt/kg, Polycisplatin exhibited a little better efficacy than cisplatin. Furthermore, mice treated with cisplatin at the dose of 1.95 mg Pt/kg exhibited severe body weight decrease by about 25%, while mice treated with Polycisplatin did not show serious body weight decrease even at its double dose of 3.9 mg Pt/kg. Furthermore, kidney indicators including kidney index, BUN, and creatinine values measured displayed that Polycisplatin is much less nephrotoxic than cisplatin. CONCLUSION: Nanoparticular Polycisplatin was successfully prepared by conjugating cisplatin to a hydrophilic polyphosphazene carrier polymer using the acid-cleavable cis-aconitic acid. Polycisplatin nanoparticles exhibit excellent tumor-targeting properties by EPR effect. The xenograft trials exhibited excellent antitumor efficacy and reduced systemic toxicity of Polycisplatin.

Design of theranostic nanomedicine (II): synthesis and physicochemical properties of a biocompatible polyphosphazene-docetaxel conjugate.

To prepare an efficient theranostic polyphosphazene-docetaxel (DTX) conjugate, a new drug delivery system was designed by grafting a multifunctional lysine ethylester (LysOEt) as a spacer group along with methoxy poly(ethylene glycol) (MPEG) to the polyphosphazene backbone ([NP]n), and then DTX was conjugated to the carrier polymer using acid-cleavable cis-aconitic acid (AA) as a linker. The resultant polyphosphazene-DTX conjugate, formulated as [NP(MPEG550)3(Lys-OEt)(AA)(DTX)]n and named "Polytaxel", exhibited high water solubility and stability by forming stable polymeric micelles as shown in its transmission electron microscopy image and dynamic light scattering measurements. Another important aspect of Polytaxel is that it can easily be labeled with various imaging agents using the lysine amino group, enabling studies on various aspects, such as its organ distribution, tumor-targeting properties, pharmacokinetics, toxicity, and excretion. The pharmacokinetics of Polytaxel was remarkably improved, with prolonged elimination half-life and enhanced area under the curve. Ex vivo imaging study of cyanine dye-labeled Polytaxel showed that intravenously injected Polytaxel is long circulating in the blood stream and selectively accumulates in tumor tissues. Polytaxel distributed in other organs was cleared from all major organs at ~6 weeks after injection. The in vitro study of DTX release from the carrier polymer showed that >95% of conjugated DTX was released at pH 5.4 over a period of 7 days. Xenograft trials of Polytaxel using nude mice against the human gastric tumor cell line MKN-28 showed complete tumor regression, with low systemic toxicity. Polytaxel is currently in preclinical study.

Design of a novel theranostic nanomedicine: synthesis and physicochemical properties of a biocompatible polyphosphazene-platinum(II) conjugate.

To develop a theranostic nanomedicine involving the antitumor-active moiety (dach)Pt(II) (dach: trans-(±)-1,2-diaminocyclohexane) of oxaliplatin (OX), a new biocompatible polyphosphazene carrier polymer was designed by grafting with a methoxy poly(ethylene glycol) (MPEG) to increase duration of circulation in the blood and with aminoethanol (AE) as a spacer group. The antitumor (dach)Pt moiety was conjugated to the carrier polymer using cis-aconitic acid (AA) as a linker, resulting in a polymer conjugate formulated as [NP(MPEG550)(AE-AA)Pt(dach)]n, named "Polyplatin" (PP). PP was found to self-assemble into very stable polymeric nanoparticles with a mean diameter of 55.1 nm and a critical aggregation concentration of 18.5 mg/L in saline. PP could easily be labeled with a fluorescence dye such as Cy5.5 for imaging studies. The time-dependent ex vivo image studies on organ distributions and clearance of Cy-labeled PP have shown that PP accumulated in the tumor with high selectivity by the enhanced permeability and retention effect but was cleared out from all the major organs including the liver in about 4 weeks postinjection. Another time-dependent bioimaging study on distribution and clearance of PP in mouse kidney using laser ablation inductively coupled plasma mass spectroscopy has shown that PP accumulates much less in kidney and is more rapidly excreted than monomeric OX, which is in accord with the very low acute toxicity of PP as shown by its high LD50 value of more than 2000 mg/kg. The pharmacokinetic study of PP has shown that it has a much longer half-life (t 1/2ß) of 13.3 hours compared with the 5.21 hours of OX and about a 20 times higher area under the curve value of 42,850.8 ng h/mL compared with the 2,320.4 ng h/mL of OX. The nude mouse xenograft trials of PP against the gastric MKN-28 tumor cell line exhibited remarkably better tumor efficacy compared with OX at the higher tolerated dose, with lower systemic toxicity.

Synthesis and properties of a new micellar polyphosphazene-platinum(II) conjugate drug.

Aiming at tumor targeting delivery of oxaliplatin using polymer therapy, a new monomeric platinum(II) complex (dach)Pt[HEDM] (dach: trans-(±)-1,2-diaminocyclohexane; HEDM: 2-hydroxyethoxydiethylmalate) was designed to include the antitumor moiety (dach)Pt and HEDM as a linker to the polyphosphazene backbone. This monomeric Pt-complex could easily be grafted to the PEGylated polyphosphazene backbone to prepare a novel polyphosphazene-Pt conjugate, [NP(MPEG550)(dach)Pt(EM)]n [MPEG550: methoxy poly(ethylene glycol) with an average molecular weight of 550; EM: ethoxymalate]. This amphiphilic polyphosphazene-Pt conjugate was found to self-assemble into stable polymeric micelles of a mean diameter of 130nm, which is suitable for passive tumor targeting by enhanced permeability and retention (EPR) effect. Pharmacokinetic study of this polymer conjugate exhibited long blood circulation as expected and longer half-life (t1/2ß=9.52h) compared with oxaliplatin (3.47h), and much larger AUC (area under the curve) value (25,831ng·h/mL) compared with oxaliplatin (1194ng·h/mL). Biodistribution study of the polymer conjugate has shown excellent tumor selectivity with the tumor to tissue ratio of 3.84 at 2h post injection and 11.7 at 24h post injection probably due to the EPR effect of the polymer conjugate while no tumor selectivity was observed for monomeric oxaliplatin. Furthermore, accumulation of this polymer conjugate in kidney was much lower compared with oxaliplatin. Also the nude mouse xenograft trial of the polymer conjugate has shown higher antitumor efficacy compared with oxaliplatin.

Preclinical evaluation of efficacy and stability of docetaxel micelle-encapsulated by a tripodal cyclotriphosphazene amphiphile.

Docetaxel formulated by micelle-encapsulation using a tripodal cyclotriphosphazene amphiphile [NP(MPEG750)(GlyPheLeu)2Et]3 (CP750) was named "Phostaxel" and compared in efficacy and stability with Taxotere(®) formulated using the surfactant polysorbate 80, which is currently in clinical use. Phostaxel has always shown better efficacy than Taxotere(®) in various xenograft trials at the same dosage and administration schedule against the tumor cell lines tested. The better efficacy of Phostaxel could be explained based on the difference in pharmacokinetic and biodistribution profiles of Phostaxel and Taxotere(®). Phostaxel exhibited significantly slower clearance rate and larger AUClast value compared with Taxotere(®). Phostaxel has also shown higher DTX distribution in tumor than Taxotere(®). In addition, Phostaxel displayed better solution stability compared with Taxotere(®) both in distilled water and in saline solution at room and refrigerator temperatures.

Synthesis and physicochemical properties of new tripodal amphiphiles bearing fatty acids as a hydrophobic group.

Saturated fatty acids (FA) were grafted using tyrosine as a spacer group to the cyclotriphosphazene ring along with equimolar hydrophilic methoxy poly(ethylene glycol) (MPEG) in cis-nongeminal way. Seven new cyclotriphosphazene amphiphiles were prepared from combinations of hydrophilic MPEGs with different molecular weights of 350, 550, 750 and 1000 and four different fatty acids of different hydrophobicity including lauric, myristic, palmitic and stearic acids. These steric amphiphiles bearing fatty acids as a hydrophobic group were found to form more stable micelles with very low critical micelle concentrations (CMC) (2.95-7.80mg/L) compared with oligopeptide analogues, and their highly hydrophobic core environment is unique and potentially useful for various biomedical applications.

Stable and efficient delivery of docetaxel by micelle-encapsulation using a tripodal cyclotriphosphazene amphiphile.

Docetaxel micelle-encapsulated by a tripodal cyclotriphosphazene amphiphilile [NP(PEG750)(GlyPheLeu)(2)Et](3) (CP750) exhibited outstanding drug-loaded micelle stability in aqueous solution compared with the polymeric micelles assembled from linear block copolymers. Furthermore, docetaxel micelle-encapsulated by CP750 is obtainable in solvent free powder form, which is immediately soluble in any aqueous media including saline and PBS and very stable to photo-degradation even in the room light at room temperature. Although docetaxel micelle-encapsulated by CP750 did not display highly improved pharmacokinetic profile compared with Taxotere currently in clinical use, its in vivo xenograft trials exhibited excellent antitumor efficacy by showing complete tumor regression against the breast cancer cells (MDA-MB-231) at a lower dose of 5mg/kg and better efficacy against gastric cancer cells (MKN-28) compared with Taxotere. Furthermore, according to the comparative acute toxicity study, toxicities associated with Taxotere may be remarkably reduced by micelle-encapsulation of docetaxel using CP750, which afforded a much higher LD(50) value of 75 mg/kg compared with 28 mg/kg of docetaxel in Taxotere. Thus docetaxel micelle-encapsulated by CP750 has entered the stage of preclinical studies.

A novel micelle-encapsulated platinum(II) anticancer agent.

A hydrophobic and water-insoluble platinum(II) compound, cis-(cha)(2)Pt(NO(3))(2) was encapsulated by macromolecular micelles self-assembled from an amphiphilic cyclotriphosphazene [NP(MPEG750)(GlyPheLeu)(2)Et](3) (CP750). The micelle-encapsulated platinum(II) compound exhibited outstanding pharmacokinetics in rats by showing long blood circulation and much larger systemic exposure (AUC=43.5 µgh/ml) compared with the free carboplatin (AUC=4.32 µgh/ml). Biodistribution study of the micellar platinum(II) compound using male Sprague-Dawley rats has shown excellent tumor to tissue ratios of 4.03 at 2h post injection and 4.67 at 24h post injection. Furthermore, the micellar platinum(II) compound exhibited more than 6 times higher cellular uptake in human cervical (HeLa) and lung (A549) tumor cells compared with the free platinum compound. Also it is surprising that the micellar platinum(II) compound displayed specifically high cytotoxicity against the stomach tumor cells (SNU638), which are one of the least responsive to chemotherapeutic agents currently in clinical use. The acute toxicity study has shown that the LD(50) values of free and the micellar cis-(cha)(2)Pt(NO(3))(2) are approximately 70 mg/kg and 90 mg/kg, respectively. Thus the platinum compound encapsulated by cyclotriphosphazene micelles is a promising candidate for preclinical studies.

Tripodal amphiphiles tunable for self-assembly to polymersomes.

Cyclotriphosphazenes grafted with equimolar amounts of a hydrophilic polyethylene glycol and a hydrophobic oligopeptide in cis-nongeminal way form a new class of tripodal amphiphiles allowing both intra- and intermolecular hydrophobic interactions that differ from linear block copolymer amphiphiles. It has been found in this study that the tripodal amphiphiles can be tuned for self-assembly from micelles to bilayered polymersomes by controlling the hydrophobicity of the oligopeptide grafted. For instance, the tripodal amphiphiles with an intermediate hydrophobicity (01) remain as stable micelles in aqueous solution. These biodegradable polymersomes exhibit outstanding physicochemical properties required for practical drug delivery and other biomedical applications. In particular, the cyclic phosphazene trimer platinated with a hydrophobic cis-bis(cyclohexylamine)Pt-moiety forms very stable polymersomes with excellent tumor selectivity by EPR effect and seems to be a promising candidate for preclinical studies.

Enzymatically degradable temperature-sensitive polypeptide as a new in-situ gelling biomaterial.

We are reporting a poly (ethylene glycol)-block-poly(alanine-co-phenyl alanine) (PEG-PAF) aqueous solution that undergoes sol-to-gel transition as the temperature increases. The sol-to-gel transition was observed at as low a concentration as 3.0-7.0 wt.%. Micellar aggregation accompanying small conformational changes of the peptide from random coils to beta-sheets is suggested as the sol-to-gel transition mechanism of the PEG-PAF aqueous solution. The PEG-PAF is stable in phosphate buffered saline, however, it degraded in the subcutaneous layer of rats. In vitro study showed that proteolytic enzymes such as cathepsin B, cathepsin C, and elastase that are present in the subcutaneous layer of the mammalian tissue might be responsible for the degradation of the polymer in rats. As a feasibility study of this material, a single shot of an aqueous insulin formulation (13.8 mg insulin/kg) showed a hypoglycemic effect over 18 days in rats. The current functional polypeptide may be very promising as an in-situ gelling system for tissue engineering, cell/stem cell therapy, and drug delivery.

A micellar prodrug of paclitaxel conjugated to cyclotriphosphazene.

A novel water soluble and biodegradable cyclotriphosphazene-paclitaxel conjugate was prepared by reacting 2'-succinyl paclitaxel with cyclotriphosphazenes bearing equimolar glycyl-L-lysine and methoxy poly(ethylene glycol) as side groups. The macromolecular conjugate was found to self-assemble in aqueous solution to form stable micelles with a mean hydrodynamic diameter of 24.7 nm and a low critical micelle concentration of 10 mg/L. The present conjugate exhibited lower than free paclitaxel but reasonably high in vitro cytotoxicity against selected human tumor cells due to their hydrolytic degradation in PBS solution.

Cellular uptake and cytotoxicity of octahedral rhenium cluster complexes.

Cellular uptake behavior of a novel class of octahedral rhenium cluster compounds, hexahydroxo complexes K(4)[{Re(6)S(8)}(OH)(6)].8H(2)O (1) and K(4)[{Re(6)Se(8)}(OH)(6)].8H(2)O (2), was evaluated in human cervical adenocarcinoma HeLa cells. Confocal microscopy and flow cytometry studies demonstrated that rhenium cluster 1 was not internalized into cell, while rhenium cluster 2 was. Conjugation of a polymer to rhenium cluster 1, namely the derivative K(4)[{Re(6)S(8)}(OH)(5)L] (3) (L is amphiphilic diblock copolymer MPEG550-CH(2)CONH-GlyPheLeuGlyPheLeu-COO(-)), considerably enhanced cellular uptake in a concentration-dependent manner and was predominantly localized in the cytoplasm and nucleus upon incubation time. The uptake of rhenium cluster 2 was mediated by energy-dependent endocytosis, whereas rhenium cluster 3 was directly ingested into cells by cell-fusion-like mechanism. According to the cytotoxicity evaluation test, both rhenium clusters 2 and 3 did not exhibit acute cytotoxic effects up to 50 microM, at the practical concentration level of biological applications. It is, therefore, expected that the rhenium cluster complexes can be promising potential candidates as diagnostic agents for medical treatment.

Nanoparticulate platinum(II) anticancer drug: synthesis and characterization of amphiphilic cyclotriphosphazene-platinum(II) conjugates.

Novel cyclotriphosphazene-platinum(II) conjugates were prepared by hydrolysis and platination of the amphiphilic cyclotriphosphazenes grafted with equimolar hydrophilic methoxy-poly(ethylene glycol) (MPEG) and hydrophobic oligopeptide. These macromolecular conjugates were found to form stable nanoparticles with a mean diameter of approximately 90-200 nm depending on the hydrophobicity of the conjugated (diamine)platinum moieties. The nanoparticulate platinum(II) conjugates have shown temperature and concentration dependent particle sizes. However, the particle sizes of the conjugates were found to decrease to a certain size as the solution concentration was decreased but remained stable even at 10 microM, which is enough for systemic delivery by injection. The conjugates exhibited lower in vitro cytotoxicity than cisplatin but reasonably good activity against selected human tumor cell lines.

Closed-loop sol-gel transition of PEG-PEC aqueous solution.

We are reporting an unusual closed-loop phase behavior of poly(ethylene glycol)-beta-poly(ethyl-2-cyanoacrylate) (PEG-PEC) aqueous solutions. As the temperature increased from 0 to 60 degrees C, the aqueous polymer solution (12 wt %) underwent sol-to-gel and gel-to-syneresis transitions. However, the polymer aqueous solution persisted as a sol phase below 4.0 wt % as well as above 16 wt % in the same temperature range, thus forming a closed-loop gel domain in the phase diagram. The closed-loop gel domain is suggested to be a result of the balance between the aggregation and the stabilization of micelles in specific temperature and concentration ranges.

A tetra(L-lysine)-grafted poly(organophosphazene) for gene delivery.

In order to develop a new gene delivery vector, a novel cationic poly(organophosphazene) was synthesized by stepwise nucleophilic substitutions of poly(dichlorophosphazene) with a hydrophilic methoxy-poly(ethylene glycol) (MPEG) as a shielding group and a branched tetra(L-lysine), LysLys(LysEt)(2), as a cationic moiety. The cationic polymer has shown to form a polyplex by DNA condensation and very low in vitro cytotoxicity probably due to the shielding effect of MPEG, which provides a basis for improving the low gene transfection yield of cationic polyphosphazenes.

Thermosensitive and biocompatible cyclotriphosphazene micelles.

A new class of thermosensitive micellar cyclotriphosphazenes has been synthesized by stepwise nucleophilic substitutions of hexachlorocyclotriphosphazene with a hydrophilic methoxy poly(ethylene glycol) (MPEG) and a hydrophobic oligopeptide selected from tetra- to hexapeptides, and characterized by means of multinuclear ((1)H, (31)P) NMR spectroscopy, elemental analysis, and dynamic light scattering (DLS) method. All the amphiphilic trimers were found to form stable micelles by self-assembly in aqueous solution and to exhibit a lower critical solution temperature in the range of 20-48 degrees C in water depending on the hydrophilic to hydrophobic balance of the side groups. The micelles formed from the trimers bearing MPEG350 and a tetra- or pentapeptide were found to have a mean diameter of 13-14 nm, while, surprisingly, the trimers bearing longer MPEG550 and hexapeptides have shown remarkable contraction of their micelle size to a mean diameter of 7-8 nm, probably due to the strong intermolecular hydrophobic interactions among the hexapeptide groups of the trimers. The local tolerance tests using rabbits have shown excellent biocompatibility of the trimers. Also a promising in vitro releasing profile was obtained for local delivery of human growth hormone (hGH) as a model protein drug.

Thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive degradable polymer.

We report a reverse thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive biodegradable polymer. The poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) aqueous solutions studied in this research underwent sol-gel-sol or sol-gel-sol-gel transition depending on the molecular weight and concentration of the polymer, whereas the corresponding disulfide multiblock copolymer aqueous solutions underwent sol-gel transition as the temperature increased in a range of 0-60 degrees C. The hydrophobic dye solubilization and dynamic light scattering of the polymer aqueous solution suggest that the poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)s undergo unimer (3 nm) to micelle (12 nm) transition, whereas the disulfide multiblock copolymers undergo unimer (6 nm) to aggregated polymer (600 nm) transition as the temperature increases. The gel duration increased from 6 h (poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)) to more than 12 days (the corresponding disulfide multiblock copolymer) in phosphate buffer saline, and the gel duration of the latter depended on the glutathione concentration of the medium. The model drug, paclitaxel, was released from the in-situ-formed poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer gel in a glutathione concentration-sensitive manner.

Thermogelling aqueous solutions of alternating multiblock copolymers of poly(L-lactic acid) and poly(ethylene glycol).

We are reporting alternating multiblock copolymers of poly(L-lactic acid)/poly(ethylene glycol) aqueous solution (> 15 wt %) undergoing sol-gel-sol transition as the temperature increases from 20 to 60 degrees C. Micelles of the multiblock copolymers (in water) are about 20 nm in radius at low temperature. They are aggregated to a larger size as the temperature increases, which should play a critical role in the sol-to-gel transition. The transition temperature and gel window were affected by the molecular weight and composition of the multiblock copolymer. In particular, the aqueous solution of an alternating multiblock copolymer (Mn approximately 6700 daltons) prepared from poly(ethylene glycol) (Mn approximately 600 daltons) and poly(L-lactic acid) (Mn approximately 1300 daltons) showed a maximum modulus at body temperature (37 degrees C). The in situ gel forming ability of the polymer aqueous solution in vivo as well as in vitro indicates that it can be a promising injectable biomaterial.

Flexibility of inorganic tennis ball structures inducing anion selectivity.

Inorganic tennis balls (ITBs), [[{Pt(betmp)(dach)}(2)Cu](2)(X)][X](3) (in which X=ClO(4) (-) (3), NO(3) (-) (4), Cl(-) (5) and Br(-) (6); dach=trans-1,2-diaminocyclohexane and betmp=bisethylthiomethylidenepropanedioate) and [[{Pt(dteym)(dach)}(2)Cu](2)(PF(6))][PF(6)](3) (7; dteym=1,3-dithiepane-2-ylidenemalonate), were prepared as crystals. Investigation of their X-ray crystal structures revealed that shapes of the cavities in ITBs show significant distortions that depend on the properties of the encapsulated anions. The CuCu* distance was observed to be longest in 7 and shortest in 5, the difference between them being 2.05 A. The flexibility of cavity structures of ITBs makes it possible to encapsulate various anions inside the cavity, while their distortions may be a reason for the difference in the encapsulating ability for anions, that is, anion selectivity. Especially, the distortions observed in 7 are so severe that the encapsulating ability of the cavity for PF(6) (-) is very low compared to other anions. The shapes of ITBs with ClO(4) (-) and BF(4) (-) ions inside their cavities are very similar; however, ClO(4) (-) is encapsulated by the cavity better than BF(4) (-), which is explicable by the difference of metal-anion interactions. This structural study on ITBs gives a clue to the origin of the anion selectivity of the cavity in ITBs previously investigated by (19)F NMR spectroscopy of the ITBs in methanol.