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Yong Joo Jun,1,* Jung Hyun Park,2,* Prakash G Avaji,1 Kyung Su Park,3 Kyung Eun Lee,3 Hwa Jeong Lee,2 Youn Soo Sohn1 1C & Pharm, Ewha Womans University, Seodaemun-gu, Seoul, Republic of Korea; 2Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seodaemun-gu, Seoul, Republic of Korea; 3Advanced Analysis Center, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: 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 ~6weeks after injection. The invitro study of DTX release from the carrier polymer showed that >95% of conjugated DTX was released at pH 5.4 over a period of 7days. 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. Keywords: docetaxel, polyphosphazene, anticancer drug, nanomedicine, theranostics

Basavaraj R Patil,1 Su Yeon Kang,2 Da Hee Jung,2 Prakash G Avaji,1 Yong Joo Jun,1 Hwa Jeong Lee,2 Youn Soo Sohn1 1C & Pharm, Ewha Womans University, Seoul 03760, Republic of Korea; 2Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Republic of KoreaCorrespondence: Youn Soo SohnC & Pharm, Ewha Womans University, Room 304, University-Industry Cooperation Building, 52 Ewhayeodae-Gil, Seodaemun-Gu, Seoul 03760, Republic of KoreaTel +82 70 7008 2120Fax +82 2 362 8813Email yssohn@ewha.ac.krHwa Jeong LeeGraduate School of Pharmaceutical Sciences, Ewha Womans University, 52 Ewhayeodae-Gil, Seodaemun-Gu, Seoul 03760, Republic of KoreaTel +82 2 3277 3409Fax +82 2 3277 2851Email hwalee@ewha.ac.krPurpose: 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.Keywords: cisplatin, polyphosphazene, platinum drug, drug delivery, nanomedicine

Docetaxel formulated by micelle-encapsulation using a tripodal cyclotriphosphazene amphiphile [NP(MPEG750)(GlyPheLeu) 2 Et] 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 AUC last 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.

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.

Prakash G Avaji,1,2,* Jung Hyun Park,1,* Hyun Jeong Lee,1 Yong Joo Jun,2 Kyung Su Park,3 Kyung Eun Lee,3 Soo-Jin Choi,4 Hwa Jeong Lee,1 Youn Soo Sohn2 1Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea; 2C & Pharm, Ewha Womans University, Seoul, Republic of Korea; 3Advanced Analysis Center, Korea Institute of Science and Technology, Seoul, Republic of Korea; 4Department of Food Science and Technology, Seoul Women’s University, Seoul, Republic of Korea *These authors contributed equally tothis work Abstract: 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 (t1/2β) 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. Keywords: polyphosphazene, anticancer agent, theranostics, nanomedicine, oxaliplatin

to glycine or glycylphenylalanine were grafted to the cyclotriphosphazene backbone bearing an equimolar hydrophilic poly(ethylene glycol) (PEG). The properties of the resultant amphiphiles were examined in comparison with those of the linear oligopeptide analogues previously reported. All cyclic phos-phazene trimers grafted with the branched tetra- and pentapeptides displayed a normal trend of thermosensitivity depending on their hydrophilic to hydrophobic balance, but the stability and particle size of their micelles were found to be greatly dependent on the fine structure of the branched oligopeptides grafted. The trimers bearing branched tetra-peptides with a low hydrophobicity were found to form unstable micelles initially, which reassemble into thermo-dynamically more stable polymersomes.

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 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.

In the search for a new thermogelling biomaterial, we are reporting poly(ethylene glycol)−poly(trimethylene carbonate) (PEG−PTMC) diblock copolymers. The PEG−PTMC (550−2750) diblock copolymer aqueous solutions (>25 wt %) underwent sol-to-gel-to-syneresis (macroscopic phase separation between the polymer and water) transition as the temperature increased. The sol-to-gel transition temperature could be controlled in a range of 20−75 °C by varying polymer concentration in water, molecular weight, and composition of the polymer. 13C NMR spectra, a transmission electron microscopic image, and dynamic light scattering suggest that the micellar aggregation through the dehydration of PEG is involved in the sol-to-gel transition. The in-situ gel formation was confirmed by subcutaneous injection of the polymer aqueous solution (30 wt %, 0.5 mL) into rats. Because of the diffusion of the polymer with a low molecular weight out of the gel, mass loss of 15 wt % was observed over 20 days in in-vivo study. On the other ...

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.

Aqueous solutions of both poly(ethylene glycol)/poly(dl-lactic acid) (PEG/PDLLA) and poly(ethylene glycol)/poly(l-lactic acid) (PEG/PLLA) multiblock copolymers underwent “clear sol-to-turbid gel-to-syneresis” transitions as the temperature increased. To investigate the stereoisomeric effect on the phase behavior, PEG/PDLLA and PEG/PLLA multiblock copolymers with a similar block length and total molecular weight were carefully synthesized. Compared with the PEG/PDLLA multiblock copolymer, the PEG/PLLA multiblock copolymer showed a lower critical gel concentration, a lower sol-to-gel transition temperature, and a wider gel window. In addition, the maximal gel modulus of the PEG/PLLA multiblock copolymer (11.0 wt % in water) was 2 times larger than that of the PEG/PDLLA multiblock copolymer. X-ray diffraction spectra, transmission electron microscopic images, and polarizing optical microscopic images suggested the amorphous state for both PEG/PDLLA and PEG/PLLA multiblock copolymer thermogels. 13C NMR spectr...

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.

New thermosensitive and micelle-forming diblock copolymers were synthesized by amide coupling of carboxylated methoxy-poly(ethylene glycols) with hydrophobic oligopeptides and characterized by means of 1H NMR spectroscopy, elemental analysis, and dynamic light scattering (DLS) measurements. Stable micelles were formed from copolymers composed of MPEG550 or MPEG750 as a hydrophilic segment and the hexapeptide, GlyPheLeuGlyPheLeuEt, as a hydrophobic segment. All the present diblock copolymers exhibited thermosensitive properties by showing a lower critical solution temperature (LCST) in water. The LCST of the copolymers composed of relatively shorter MPEG350 and GlyPheLeuAspEt2 was observed at much higher temperature (48 °C) compared with the LCST (29 °C) of the cyclotriphosphazene analogues bearing the same hydrophilic and hydrophobic segments as side groups. The remarkably lower LCST of the cyclotriphosphazene analogue is presumed to be due to the structural effect of the cis-nongeminal conformation of its three hydrophobic oligopeptides, but such a structural effect was found to diminish as the chain lengths of the hydrophilic and hydrophobic blocks of the copolymers increased.

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 °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.

We reported aqueous solutions of poly(caprolactone-b-ethylene glycol-b-caprolactone) (PCL−PEG−PCL) that underwent sol−gel−sol transition as the temperature increased (Macromolecules 2005, 38, 5260−5265). However, when the triblock copolymer aqueous solution (20 wt %), initially as a sol phase, was left at room temperature (20 °C), it turned into an opaque gel in 1 h. The crystallization of the PCL−PEG−PCL triblock copolymer in water was suggested to be responsible for such a kinetic aspect of the phase transition. In addition, PEG/PCL multiblock copolymers were synthesized by coupling the triblock copolymers using terephthaloyl chloride. Even though both PCL−PEG−PCL triblock and PEG/PCL multiblock copolymer aqueous solutions (20 wt %) instantaneously undergo a sol-to-gel transition upon injection into 37 °C water and their thermogels show a maximum modulus at around body temperature (35−42 °C), the multiblock copolymer shows a pronounced sol phase stability at room temperature. The fundamental difference ...

Biocompatible and thermosensitive poly(organophosphazenes) with a lower critical solution temperature (LCST) below body temperature have been designed with the aim for the local delivery of peptide and protein drugs. These polymers could be synthesized by introducing short chain tri- or tetraethylene glycol as a hydrophilic group and a dipeptide, GlyGluEt2 as a hydrophobic group into the polyphosphazene backbone. The local tolerance tests using rabbits have shown that our polymers are biocompatible. Using the amphiphilic properties of these polymers, in vitro studies were performed for loading and releasing of a human growth hormone (hGH) as a model drug. The entrapment efficiency (%) of hGH by the polymer decreased as its polymer concentration increased, but exhibited high efficiency of more than 95% even at 20% hGH concentration in the polymer. The entrapped hGH has shown to be controlled releasing for 3-4 days.

A new biodegradable water-soluble phosphazene trimer–doxorubicin conjugate was synthesized, in which equimolar hydrophilic methoxy-poly(ethylene glycol) with a molecular weight of 350 (MPEG350) and a tumor-specific tetrapeptide (Gly-Phe-Leu-Gly) were grafted to cyclotriphosphazene. The present conjugate exhibited cytotoxicity lower than that of free doxorubicin (IC50 = 0,10 μM) but a reasonably higher in vitro cytotoxicity (IC50 = 1.1 μM) against the leukemia L1210 cell line probably due to its enzymatically controlled release.

A new amphiphilic poly(organophosphazene) was synthesized by stepwise nucleophilic substitutions with a hydrophilic methoxy poly(ethylene glycol) with an average molecular weight of 350 (MPEG350) and a hydrophobic glycyl- l -glutamate as side groups, and then an antitumor (dach)platinum(II) (dach: trans -(±)-1,2-diaminocyclohexane) moiety was conjugated to the polymer using the dipeptide as a spacer. This polymeric platinum conjugate was found to be accumulated in the tumor tissue to a remarkably greater extent than in the normal tissue (tumor/tissue ratio >4), probably due to the excellent EPR effect and the long circulating properties of the polymer conjugate ( t 1/2β = 6.2 h and AUC = 4020 nmol h/ml) compared with carboplatin ( t 1/2β = 0.42 h and AUC = 120 nmol h/ml). The polymer conjugate also exhibited high in vitro cytotoxicity comparable to cisplatin against several human tumor cells tested.

A new class of thermogelling poly(organophosphazenes) bearing a hydrophilic methoxypoly(ethylene glycol) (MPEG) and a hydrophobic tri or tetrapeptide such as GlyPheLeuEt, GlyPheIleEt, GlyLeuPheEt, and GlyPheLeuGlyEt have been synthesized and characterized by means of multinuclear (1H, 31P) NMR spectroscopy, gel permeation chromatography, viscometry, and elemental analysis. The gelation of the present polymers is presumed to be attributed to the intermolecular interaction between the hydrophobic oligopeptide side groups, which can form strong physical junction zones in the polymer aqueous solution. The gelation properties of the polymer were affected by the subtle change in the nature of the hydrophobic oligopeptide, composition of the hydrophilic to hydrophobic side groups, and the concentration of the polymer solutions. Among the present thermogels, the copolymer with equimolar MPEG and GlyPheIleEt as side groups showed the excellent gel phase persisting over 35–43 °C, which indicates that it is a new promising material for drug delivery and tissue engineering.

Poly(caprolactone-b-ethylene glycol-b-caprolactone) (PCL−PEG−PCL) triblock copolymer aqueous solution (>15 wt %) undergoes the sol−gel−sol transition as the temperature increases from 10 to 60 °C. The mechanism and structure−property relationship of the sol−gel transition were investigated. In particular, compared with the PEG−PCL−PEG triblock copolymers recently reported by our group, PCL−PEG−PCL has (1) a synthetic advantage without a hexamethylene diisocyanate coupling step, (2) a wider gel window of over 15−32 wt %, and (3) a larger gel modulus. Both PEG−PCL−PEG and PCL−PEG−PCL polymers are an important progress in the biodegradable thermogelling system in that they can be lyophilized in a powder form, are easy to handle, are easy to redissolve to a clear solution, and show little syneresis through the gel phase.

Mono(trifluoroacetato)platinum(IV) complexes of the formula [PtIV(dach)L3(TFAc)] (dach = trans-(±)-1,2-diaminocyclohexane, TFAc = trifluoroacetate, L = acetate or propionate) could be prepared from the reaction of [PtIV(dach)L3(OH)] with trifluoroacetic anhydride in the presence of a base and the crystal structure of compound 4 was determined by X-ray crystallography. In vitro antitumor activity of complex 4 (ED50 = 3.1 μM) was found to be much higher than carboplatin (ED50 = 10.2 μM).

Nanosized water soluble poly(organophosphazenes) were synthesized by grafting hydrophilic methoxy poly(ethylene glycols) and hydrophobic dipeptide ethyl esters as side groups to the phosphazene backbone. Their hydrodynamic volume could be controlled in the range of 10–30 nm in diameter depending on the length of the side groups and the molecular weight of the polymers. These polymers exhibited a lower critical solution temperature in the range of 60–105 °C and hydrolytic degradability, which can afford applications to a variety of drug delivery systems. The hydrolytic properties of the present poly(organophosphazenes) have been studied at 37 °C in different pH buffer solutions by means of gel permeation chromatography. The polymers substituted with the more hydrophobic and more bulky dipeptide groups caused slower hydrolysis and the polymer hydrolysis occurred more rapidly in the acidic buffer solution than in the neutral and basic solutions.

A new series of hematoporphyrin-platinum(II) conjugates was prepared by platination of the glutamate ligand tethered to hydrophilic hematoporphyrin derivatives, in which different numbers of ethylene oxide unit were introduced to modulate the hydrophobic/hydrophilic balance of the conjugates. The antitumor activity of the hematoporphyrin-platinum(II) conjugates was assayed in vitro and in vivo against the leukemia L1210 cell line. Among the complexes, compound 11 exhibited not only higher in vivo activity (T/C% = 192) than cisplatin (T/C% = 184) and carboplatin (T/C% = 168), but also elevated tumor-localizing effect (tumor/muscle ratio > 3).

We report a thermosensitive polypeptide consisting of hydrophilic poly(ethylene glycol)s and hydrophobic phenyl alanine ethyl esters. It is soluble in most solvents and undergoes a micelle (radius10 nm) to nanoparticle (radius100 nm) transition in water as the temperature increases. The transition temperature could be controlled to between 30 and 40°C by varying the number of poly(ethylene glycol) grafts. This system might be promising for advanced drug delivery.

The lower critical solution temperature (LCST) of cyclotriphosphazenes and polyphosphazenes grafted with a hydrophilic poly(ethylene glycol) (PEG) and a hydrophobic amino acid ester (AAE) as side groups could be quantitatively correlated with the overall hydrophobicity given by linear combination of the logarithmic partition coefficients of the corresponding free PEG and AAE measured in the 1-octanol/water system. Thus the LCSTs of phosphazenes were found to be well described as a linear function of a new parameter Pt defined as linear combination of the logarithmic partition coefficients of the free side groups, Kpeg and Kaae: P t =x log K peg +(2−x) log K aae where x is the mole fraction of PEG. The LCST of cyclotriphosphazenes has shown to be more sensitive to change in hydrophobicity than polyphosphazenes, indicating that the nature of the molecular structure plays an important role in determining their LCST. The additivity of the logarithms of the two partition coefficients for the parameter may indicate that the contribution of each constituent is independent, providing a facile method for prediction of the LCST of thermosensitive polymers.

The trans-(+/-)-1,2-diaminocyclohexaneplatinum(II) complexes of multidentate L-glutamate (Glu) and L-aspartate (Asp) were prepared and their antitumor activity was examined in relation with their coordination modes. All these complexes were obtained as a mixture of (O,O')- and (O,N)-chelate isomers due to rapid isomerization of the initially formed (O,O')-isomer to the thermodynamically more stable (O,N)-isomer. The (O,O')/(O,N)-isomeric mixture with the mole ratio of 80/20 exhibited excellent antitumor activity while the pure (O,N)-isomer was only marginally active. Therefore, in order to prevent the linkage isomerization of the active (O,O')-isomer to the inactive (O,N)-isomer, we have designed N-substituted amino dicarboxylic acids as a leaving group and prepared a new series of complexes, [Pt(dach)(RGlu)] and [Pt(dach)(RAsp)] (dach=trans-(+/-)-1,2-diaminocyclohexane; R=acetyl (Ac), propionyl (Pro), pivaloyl (Piv), carbobenzyloxy (Cbz) or phthaloyl (Phth)) and characterized by means of elemental analyses, and 1H NMR, 195Pt NMR and IR spectroscopies. The N-substituted amino dicarboxylate ligands were found to coordinate to platinum(II) ion through only the (O,O')-chelation mode, and their Pt(II) complexes were chemically stable in aqueous solution. The present Pt(II) complexes of N-substituted amino dicarboxylic acids showed excellent antitumor activity against both murine leukemia L1210 and human tumor cells. Especially, the highly hydrophobic N-phthaloylglutamate complex, [Pt(dach)(PhthGlu)], exhibited an outstanding in vitro activity (IC50=2.22 microM) on the human stomach cancer cells which are not responsive to cisplatin and carboplatin.

The antitumor platinum(II) compound, [Pt(dach)(Glu)] (dach= trans (±)-1,2-diaminocyclohexane, Glu=glutamate) was formulated with a stealth liposome to improve its biological activity. Liposomes were composed of PC/PEG2000-PE/CH (PC=1,2-diacyl-glycero-3-phosphocholine; PEG2000-PE=poly(ethylene glycol)2000-1,2-diacyl-glycero-3-phosphoethanolamine; CH=cholesterol) involving different acyl moieties of phospholipids such as DO (dioleoyl), DM (dimyristoyl) or DS (distearoyl) group. Among the different acyl groups in the stealth liposomes, the DM formulation was optimal for the preparation of the liposomal [Pt(dach)(Glu)] at the mole ratio of DMPC/PEG2000-DMPE/CH=50/5/45 and at the weight ratio of drug/lipid=1/20, which is represented as l -[Pt(dach)(Glu)]. In vitro cytotoxicity was examined in sensitive A2780 and ME180 and their cisplatin-resistant A2780/PDD and ME180/PDD cancer cells. l -[Pt(dach)(Glu)] was 2∼3 times more cytotoxic than the free complex [Pt(dach)(Glu)] and cisplatin in sensitive cells, and 4∼8 times more cytotoxic in resistant cells. Thus, the resistance index of l -[Pt(dach)(Glu)] was 1.3∼2 while those of the free complex and cisplatin were 5∼6, which indicates that l -[Pt(dach)(Glu)] overcome the cisplatin resistance in both resistant cells. In vivo antitumor activity was assayed against the L1210/S leukemia. The optimal activities (% T/C) of the free complex and l -[Pt(dach)(Glu)] were >459/20 and >442/200 mg/kg, respectively. Considering the amount of the platinum complex in l -[Pt(dach)(Glu)], the liposomal [Pt(dach)(Glu)] displayed 2-fold higher drug potency than the free complex. The biodistribution experiment using LE52 tumor-bearing mouse showed excellent lung targeting property of l -[Pt(dach)(Glu)].

The aqueous solution of poly(lactic acid-co-glycolic acid)-g-poly(ethylene glycol) becomes a gel as the temperature increases. The sol-to-gel transition temperature can be controlled from 15 to 45 °C by varying the number of poly(ethylene glycol) grafts and the composition of the polymer. In addition, hysteresis between heating and cooling cycles could be controlled by adding poly(ethylene glycol) with different molecular weights as an additive. To prove the hypothesis of micellar aggregation for the sol-to-gel transition and the change in hydration status for the gel-to-sol transition, several experiments were performed. Small-angle neutron scattering and Raman spectroscopy sensitively detected the sol-to-gel transition, because it involves aggregation of the scattering particle of micelles. IR and 13C NMR showed that little change in hydration status is involved during the sol-to-gel transition, whereas significant change in hydration status is involved in the gel-to-sol transition. The intrinsic viscos...

Thermosensitive cyclotriphosphazenes bearing alkoxy poly(ethylene glycol) and amino acid esters as side groups could be functionalized to chelate the antitumor (diamine)platinum(II) moiety through the dicarboxylate group of the amino acid substituent on the cyclic phosphazene ring. Surprisingly, like the precursor cyclotriphosphazenes, these (diamine)platinum(II)–cyclotriphosphazene conjugates were also found to exhibit variable lower critical solution temperatures (LCST) in the wide range of 12 to 92 °C. Furthermore, the present conjugates have shown outstanding in vitro and in vivo antitumor activities due to controlled release of the antitumor (diamine)platinum(II) moiety with hydrolytic degradation of the phosphazene ring. A few of these conjugates have shown LCSTs below body temperature, and it has been shown from a model animal experiment that the conjugates with a LCST below body temperature may be applied to local drug delivery by direct intratumoral injection.

A new series of DNA binding 5,10,15-tri(N-methyl-4-pyridiniumyl)porphyrin (TrisMPyP)-platinum(II) conjugates was synthesized, in which different spacer ligands were used for appropriate coordination to platinum(II) complexes. Compound 9b exhibited in vivo antitumor activity (T/C%, 294) superior to cisplatin (T/C%, 184) against the leukemia L1210 cell line.

One-dimensional (1-D), two-dimensional (2-D), and three-dimensional (3-D) coordination polymers were prepared by self-assembly of binary metal complex systems, copper(II) nitrate and (en)Pt(II)(nic)(2) or (dmpda)Pt(II)(isonic)(2) (en = ethylenediamine, dmpda = 2,2'-dimethyl-1,3-propanediamine, nic = nicotinate, and isonic = isonicotinate), in aqueous solutions. Equimolar reactions of copper(II) nitrate with (dmpda)Pt(II)(isonic)(2) and (en)Pt(II)(nic)(2) resulted in 1-D ([(dmpda)Pt(isonic)(2)Cu(OH(2))(3)](NO(3))(2))(n)() (1) and 2-D ([(en)Pt(nic)(2)Cu(OH(2))](NO(3))(2))(n) (2), respectively, but the reaction of (en)Pt(II)(nic)(2) with excess copper(II) nitrate gave 3-D ([((en)Pt(nic)(2))(3)Cu(5)(OH)(2)(OH(2))(6)](NO(3))(8))(n) (3). The local structure of crystal 1 has a mononuclear copper unit, 2 has a dinuclear copper unit with a Cu-Cu distance of 2.659(5) A, and 3 has a pentanuclear copper unit. The methyl groups of the dmpda ligand are located in the space between two isonicotinate ligands of 1, which is presumed to be an important factor to determine the final structure of the crystal formed by self-assembly. Magnetic behaviors of crystals 1-3 examined in the temperature range of 4-300 K appear to be governed by the local structures around the copper(II) ions and do not indicate any significant long-range magnetic exchange interactions along the polymeric chain.

For effective administration of lipophilic trans(+/-)-1,2-diaminocyclohexaneplatinum(II) complexes of malonate derivatives [(dach)PtL, L=allylmalonate (AM), diallylmalonate (DAM), allylbenzylmalonate (ABM), or dibenzylmalonate (DBM)] in aqueous solution, we have applied three different liposome formulations and evaluated their physical and chemical properties, along with their in vitro cytotoxicity. The liposome formulations were composed of DMPC / DMPG [DMPC=1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPG=1,2-dimyristoyl-sn-glycero-3-(phospho-rac-1-glycerol) (sodium salt)] in different molar ratios (7/3 or 3/7) or an equimolar DOTAP/DOPE formulation (DOTAP=1,2-dioleoyl-3-trimethylammonium propane, DOPE=1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). Preliposomal powders of the platinum complexes were prepared by lyophilization, and reconstituted in aqueous solution to obtain the final liposomal platinum complexes. Due to the lipophilicity of the malonatoplatinum complexes, the entrapment efficiency of drugs within the liposomes was over 90% except for the AM complex, and platinum drug stability was also satisfactory (>90%) in these liposomal systems. In vitro cytotoxicity was tested in human ovarian carcinoma cells sensitive (A2780) and resistant to cisplatin (A2780/PDD). In both cell lines, the liposomal DBM complex was much more cytotoxic than the corresponding DAM and ABM complexes, which means that the more hydrophobic benzyl substituent affords higher cytotoxicity than the allyl substituent in the malonato leaving group. Furthermore, the DBM complex in DMPC/DMPG formulations was effective against both sensitive and resistant A2780 cells (resistance indexes (RI)=1.10-1.49), showing lack of cross-resistance to cisplatin. Therefore, the liposomal DBM complex in the DMPC/DMPG formulations is a promising candidate for stable pharmaceutical liposomal platinum complexes.

The reaction of [Pt(IV)(dach)(OH) 4 ] (dach= trans -(±)-1,2-diaminocyclohexane) with an excess of carboxylic acid at room temperature afforded tricarboxylatoplatinum(IV) complexes, [Pt(dach)(L) 3 (OH)] (L=acetato, propionato, valerato) in contrast to the carboxylation reaction with excess carboxylic anhydrides, which gave only tetracarboxylatoplatinum(IV) complexes as a major product. The protonation on hydroxo ligand of the platinum(IV) complex to form aqua ligand seems to be followed by nucleophilic substitution by carboxylate or chloride anion. Such nucleophilic substitution afforded only partially substituted tricarboxylatoplatinum(IV) complexes in carboxylic acid solvent whereas the reaction in 0.1 M HCl solution gave tetrachloroplatinum(IV) complex. This difference is explainable by different p K a values of the four aqua ligands, among which the last aqua ligand may have sufficiently low p K a value allowing the protonation only in strong acidic media like hydrochloric acid.

higher content of MPEG and shorter chain length of alkylamines of the polymers afforded the higher LCST. The LCSTs of the polymers exhibited almost concentration-independent behavior in the range of 3-30 wt % of the polymers in aqueous solutions. The polymers showed the higher LCSTs in the acidic solutions than in the neutral and basic solutions. The ionic strength of the polymer solution affected the LCST, which decreased with increased NaCl concentration. The polymer bearing almost equimolar substitutuents with the -N-P-N- unit has shown the LCST more sensitive to NaCl and pH than that with the -N-P-O- unit. The polymers were found to degrade in acidic solution but be very stable in alkali solution as well as in the buffer solution of pH 7.4.

Thermosensitive poly(organophosphazenes) bearing α-amino-ω-methoxy-PEG (AMPEG) and hydrophobic l-isoleucine ethyl ester (IleOEt) as side groups have been synthesized, and their reversible sol−gel p...

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 (t1/2β=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.

A new series of quinolone-platinum(II) conjugates, [Pt(Q'-NH2)(dmso)X2] and cis-[Pt(Q"-en)X2], where Q' and Q" are quinolones (flumequine, nalidixic acid or oxolinic acid) linked to monodentate and bidentate amine ligands, respectively, and X2 is Cl2 or 1,1-cyclobutanedicarboxylate, have been synthesized with the aim of examining the synergetic antitumor activity of quinolone intercalation and platinum(II) chelation. The complexes were characterized by elemental analyses and IR and multinuclear (1H and 195Pt) NMR spectroscopies, and then subjected to in vitro and in vivo bioassays using the leukemia L1210 cell line.

The solvent effect on the lower critical solution temperature (LCST) of poly(organophosphazenes) with methoxy-poly(ethylene glycol) (MPEG) and amino acid esters as side groups was examined in terms of the structure of polyphosphazenes in aqueous solutions containing one of the organic solvents selected from monoalcohols, ethylene glycol derivatives, alkylamines, and other common solvents. When such a solvent was added to the aqueous solutions of the polymers, their LCST was found to be mainly dependent on the hydrophobic and hydrophilic properties of the solvents. Most of the alcohols and amines with shorter alkyl chains increased the LCST of the polymers but those with longer chains decreased the LCST. Trifluoroethanol (TFE) showed a strong LCST decreasing effect in spite of its short chain, which seems to be due to its strong hydrophobicity. Temperature-induced molecular weight fractionation of the polymer bearing MPEG350 (M w= 350) and L-aspartic acid ethyl ester as a side group was carried out by using the LCST decreasing effect of TFE, and the fractionated samples were characterized by gel permeation chromatography (GPC) and 1H- and 31P NMR spectroscopies. Thus it has been shown that a polymer may be fractionated to the higher and lower molecular weight fractions with smaller polydispersity indices (PDI): the polymer with the weight-average molecular weight (M w) of 73,500 with PDI of 5.56 was fractionated to those of 106,000 with PDI of 4.37 and 11,000 with PDI of 1.86.

The surfactant effect on the lower critical solution temperature (LCST) of thermosensitive poly(organophosphazenes) with methoxy-poly(ethylene glycol) and amino acid esters as side groups was examined in terms of molecular interactions between the polyphosphazenes and surfactants including various anionic, cationic, and nonionic surfactants in aqueous solution. Most of the anionic and cationic surfactants increased the LCST of the polymers: the LCST increased more sharply with increasing length and hydrophobicity of the hydrophobic part of the surfactant molecule. The ΔLCSTs (T0.03M − T0M), the change in the LCST by addition of 0 and 0.03 M sodium dodecyl sulfate (SDS), were found to be 7.0 and 14.5 °C for the polymers bearing ethyl esters of glycine and aspartic acid, respectively. The LCST increase of poly(organophosphazene) having a more hydrophobic aspartic acid ethyl ester was 2 times larger compared with that of the polymer having glycine ethyl ester as a side group. The binding behavior of SDS to the polymer bearing glycine ethyl ester as a hydrophobic group was explained from the results of titration of the polymer solutions containing SDS with tetrapropylammonium bromide. Graphic models for the molecular interactions of polymer/surfactant and polymer/surfactant/salt in aqueous solutions were proposed.

Pale blue crystals were obtained from an aqueous solution containing equimolar amounts of (dmpda)Pt(N-nic) 2 (dmpda=2,2-dimethyl-1,3-propanediamine, nic=nicotinate) and copper(II) nitrate and subjected to X-ray crystallography, which revealed that the crystals are a composite material of the formula [(dmpda)Pt(N-nic) 2 ] 2 ·Cu(OH 2 ) 5 ·2NO 3 ·4.5H 2 O. Copper(II) nitrate was found to be intercalated between the platinum complex layers which were constructed by non-covalent bonding interactions such as hydrogen bonding, π–π* stacking and electrostatic interactions of the platinum(II) complex. A linear array of the intercalated cationic Cu(OH 2 ) 5 complex was observed. The value of the effective magnetic moment of the composite material is subnormal ( μ eff =1.57 BM) at room temperature.

The antitumor platinum moiety, (dach)Pt2+ (dach = trans(±)-1,2-diaminocyclohexane), was introduced to a poly(organophosphazene) using dicarboxylate spacers, glutamate or aspartate. After characterization of the polymeric conjugates by means of multinuclear (1H, 31P) NMR spectroscopy, elemental analysis, and gel-permeation chromatography (GPC), their in vitro hydrolytic behavior was examined in buffer solutions at different pHs and temperatures by monitoring with GPC. Hydrolytic properties of the conjugates were dependent on pH and temperature of the polymer solutions, and their degradation occurred more rapidly at lower pH and higher temperature. The antitumor activity of the conjugates was evaluated both in vitro and in vivo against the murine leukemia L1210 cell line and found to be dependent on the structures of the conjugates, and some of them exhibited higher in vivo activity than cisplatin and carboplatin.

New platinum(II) complexes A2Pt(ABM) (A=triethylphosphine (TEP); A2=2,2-dimethyl-1,3-propanediamine (DMPDA), trans-(±)-1,2-diaminocyclohexane (DACH); ABM=allylbenzylmalonate) have been synthesized in aqueous solution and characterized by means of X-ray analysis and multinuclear NMR spectroscopy. The crystal structures of cis-(TEP)2Pt(ABM) (monoclinic P21/n, a=12.179(3), b=16.869(7), c=12.870(3) A, β=93.27(2)°, V=2757(1) A3, Z=4, R=0.0390) and (DACH)Pt(ABM)·2H2O (triclinic P1, a=7.649(3), b=14.064(2), c=19.190(8) A, α=98.98(2), β=90.05(3), γ=105.73(2)°, V=1961(1) A3, Z=2, R=0.0455) have been solved. The platinum atom in both complexes adopts a typical square planar arrangement with each coligand in cis positions. In the solid state, the ABM ligand exhibits different chelation modes depending on the coligands: (O,O′)-chelation in the phosphine analog and (O,alkene)-chelation in the amine analogs. For the phosphine complex, the (O,O′)-chelation mode of the ABM ligand is impregnable even in solutions at variable temperatures, probably due to the strong π-bonding between the phosphorus and platinum atoms. However, (diamine)platinum(II) complexes have shown interesting linkage isomerism between the (O,O′)- and (O,alkene)-chelation modes in solutions depending on solvent and temperature.

The hydrolytic properties of the novel biodegradable thermosensitive poly(organophosphazenes) with methoxypoly(ethylene glycol) (MPEG) and amino acid esters as side groups have been studied by means of gel permeation chromatography and 31P and 1H NMR spectroscopy and by identification of the hydrolysis products. The polymers substituted with α-amino acid esters were hydrolyzed faster than that with β-amino acid ester. The higher content of the amino acid ester in the polymer backbone caused enhanced hydrolysis. The rate of the polymer degradation decreased in the order of methyl > ethyl > benzyl esters. The polymer hydrolysis occurred more rapidly in both acidic and basic buffer solutions than in the neutral solution. The 31P NMR spectra of the polymers with high content of glycine ethyl ester showed that the polyphosphazene backbone underwent fragmentation mostly to small molecules after incubation in the buffer solution of pH 10 for 26 days. Phosphates and ammonia were formed as hydrolysis products in m...

New cyclotriphosphazenes fully substituted with dicarboxylic amino acid esters (aminomalonate, aspartate and glutamate) were synthesized and characterized by means of elemental analysis, IR and multinuclear (1H, 13C and 31P) NMR spectroscopies, and X-ray crystallography. The rate of full substitution of hexachlorocyclotriphosphazene with the amino acid esters has been found to be largely dependent on the steric hindrance of the corresponding amino acid esters. The full substitution of the chlorotrimer with aminomalonate was completed in 6 h when the reactants were refluxed in tetrahydrofuran (THF), but the full substitution with aspartate or glutamate required more severe conditions: The substitution with aspartate was completed in 12 h by refluxing the reactants in the presence of n-Bu4NBr as the catalyst in toluene, but the substitution with glutamate could not be completed even after refluxing for more than two days in the presence of the same catalyst in toluene. To endow the above compounds with the functionality for incorporation of metal moiety, they have been hydrolyzed in aqueous solutions of alkali metal hydroxides to yield the corresponding metal salts, which can afford twelve active sites.