Structural analysis of human sterol transfer protein STARD4.

The steroidogenic acute regulatory protein (StAR)-related lipid transfer domain-4 (STARD4) is a sterol-binding protein that is involved in cholesterol homeostasis by intracellular sterol transport. In this work, we determined the crystal structures of human STARD4 and its Ω1-loop mutant in apo forms at 1.95 and 1.7 Šresolutions, respectively. The structure of human STARD4 displays a conserved α-helix/ß-grip fold containing a deep hydrophobic pocket. The Ω1-loop which serves as a lid for the hydrophobic pocket has a closed conformation. The shape of the sterol-binding cavity in the closed form is not complementary to accommodate cholesterol, suggesting that a conformational change of the Ω1-loop is essential for sterol binding. The human STARD4 displayed sterol transfer activity between liposomes, and the mutations in the Ω1-loop and the hydrophobic wall abolished the transfer activity. This study confirms the structural conservation of the STARD4 subfamily proteins and the flexibility of the Ω1-loop and helix α4 required for sterol transport.

Neutrophil membrane-coated therapeutic liposomes for targeted treatment in acute lung injury.

Acute lung injury (ALI) is one of the most common comorbidities associated with sepsis and can lead to acute respiratory distress syndrome. Intense inflammatory response due to excessive activation and uncontrolled infiltration of neutrophils are the central processes in the development of sepsis-induced ALI. In this study, a biomimetic nanoplatform that is a neutrophil membrane-coated liposome-loaded acidic fibroblast growth factor (aFGF@NMLs), which can selectively target the inflamed lung and effectively alleviate sepsis-induced ALI via inflammation suppression, was constructed. In vitro findings revealed that aFGF@NMLs has pro-inflammatory cytokine binding capabilities and can promote cellular uptake, substantially attenuate inflammatory responses, and enhance cellular antioxidant capacity. The in vivo results show that aFGF@NMLs can specifically accumulate in injured lungs in ALI mice after intravenous injection, thereby reducing the secretion of pro-inflammatory cytokines, inhibiting pulmonary cell apoptosis, and promoting lung function recovery. In conclusion, aFGF@NMLs demonstrated anti-inflammatory effects, mitigated the progression of ALI, and contributed to the disease prognosis. This research offers an innovative strategy and concept for the clinical treatment of diseases related to pulmonary inflammation.

Synthesis, anticancer activity and potential application of diosgenin modified cancer chemotherapeutic agent cytarabine.

Diosgenin (DG), a steroidal saponin, is mainly found in yam tubers. DG and its derivatives displayed significant pharmacological activities against inflammatory, hyperlipidemia, and various cancers. DG was selected to modify the cancer chemotherapeutic agent cytarabine (Ara-C) due to its anti-tumor activities as well as lipophilicity. After characterization, the biomembrane affinity and the kinetic thermal processes of the obtained DG-Ara-C conjugate were evaluated by differential scanning calorimetry (DSC). Thin hydration method with sonication was applied to prepare the DG-Ara-C liposomes without cholesterol since the DG moiety has the similar basic structure with cholesterol with more advantages. Dynamic Light Scattering (DLS) analysis and cytotoxic analysis were employed to characterize the DG-Ara-C liposomes and investigate their biological activities, respectively. The results indicated that DG changed the biomembrane affinity of Ara-C and successfully replaced the cholesterol during the liposome preparation. The DG-Ara-C liposomes have an average particle size of around 116 nm with a narrow size distribution and revealed better anti-cancer activity against leukemia cells and solid tumor cells than that of free DG or Ara-C. Therefore, it can be concluded that DG displayed the potential application as an anti-cancer drug carrier to improve the bio-activities, since DG counted for a critical component in modulating the biomembrane affinity, preparation of liposome, and release of hydrophilic Ara-C from lipid vesicles.

Dual cross-linking systems of functionally photo-cross-linkable and thermoresponsive polyphosphazene hydrogels for biomedical applications.

Photo-cross-linkable, functionalized, and thermosensitive polyphosphazenes were synthesized to develop a dual cross-linking system with properties of mechanically suitable strength and controllable biodegradation for injectable biomedical applications. The aqueous solutions of the polymers exhibited sol-gel transition behaviors against temperature. The incorporated methacrylate groups were photo-cross-linked upon UV light under mild conditions, which resulted in the formation of compact three-dimensional networks. The thermoresponsive hydrophobic interactions at body temperature facilitated the rapid dual cross-linking accomplishment of the photo-cross-linking even under mild conditions. The characteristics of the polymers such as pore size and density showed that the inner three-dimensional networks depended on the degree of cross-linking of methacrylate units. Mechanical properties of the gel were also improved several folds after developing the photo-cross-linking in the network from the in vivo degradation studies. The results demonstrate that the photo-cross-linkable and thermoresponsive polyphosphazenes have great potential as injectable, biodegradable, and controllable carriers for various biomedical applications by tuning the mechanical gel property and the degradation rate.

Synthesis and biological evaluation of arginyl-diosgenin conjugate as a potential bone tissue engineering agent.

Water-soluble arginyl-diosgenin (Arg-DG) conjugate was designed, synthesized, and evaluated for a biological activity. The Arg-DG conjugate was characterized using FT-IR, 1 H NMR, 13 C NMR, and HPLC-MS analyses, followed by a biological activity evaluation. Compared with DG, the Arg-DG conjugate showed a decreased cytotoxicity against L929 cells and an increased antiproliferative activity against hepatocellular cells. The safety of the Arg-DG conjugate was confirmed using the highly sensitive Alamar Blue assay, which indicated that it increased the cellular metabolic activity at suitable concentrations. The Arg-DG conjugate promoted an endothelial tube formation as well. Furthermore, the Arg-DG conjugate improved the bone morphogenetic protein 2 (BMP2)-induced osteoblastic differentiation with synergistic effects on alkaline phosphatase (ALP) activity and mineralization. These results suggest that the Arg-DG conjugate developed in this study has great potentials for biomedical applications such as bone tissue engineering.

Injectable poly(organophosphazene)-camptothecin conjugate hydrogels: synthesis, characterization, and antitumor activities.

The objective of this study is to develop an effective polymer therapeutics involving camptothecin (CPT) with enhanced efficacy and lessened systemic side-toxicity for cancer treatment. Polymer-CPT conjugates (PCCs), which consisted of CPT-20-glycinate and poly(organophosphazene) bearing carboxylic acid, were synthesized, characterized for physicochemical properties, in vitro degradation and CPT release behaviors from the PCC, and evaluated their anticancer activity. The aqueous solutions of all these PCCs showed a thermo-responsive sol-gel transition behavior for injectable application near room temperature. The CPT incorporated into the hydrogel was proven to be stable in vitro over 15 days. The in vitro cytotoxicity of the PCC was verified to be effective against four kinds of human cancer cell lines. The in vivo anticancer activity study with HT-29 colon cancer cell xenografted mice showed that the intratumorally injected PCC hydrogel inhibited the tumor growth more effectively relative to CPT alone (-29% vs. 130% in tumor size).

MRI-monitored long-term therapeutic hydrogel system for brain tumors without surgical resection.

To overcome the unresolved issues of conventional therapeutic approaches such as radiation therapy, chemotherapy, combinational chemotherapy, and surgical treatment, we designed an injectable 'MRI-monitored long-term therapeutic hydrogel (MLTH)' system as an alternative/adjuvant approach for brain tumors. The MLTH system consists of a thermosensitive/magnetic poly(organophosphazene) hydrogel (the magnetic hydrogel) as a biodegradable imaging platform and an anticancer drug as a therapeutic agent via a simple physical mixing. The MLTH system has adequate properties for the MRI-monitored long-term therapy as follows: injectability, localizability due to fast gelation at body temperature, biocompatibility, biodegradability, sustained drug release, and MR imaging function. Since the MLTH system only requires a very small-sized pin hole injected into the area of brain tumors stereotactically, we suggest that the MLTH system can be an alternative/adjuvant approach to treat the malignant brain tumors without any surgical resection. Furthermore, we expect that the MLTH system can minimize the side effects from either an intravenous injection or surgical operation because one of the aims of MLTH is to focus on the sustained local delivery of anticancer drugs via a one- or two-time intratumoral injections. Thus, we assessed successfully the MRI-monitored long-term therapeutic potentialities of the MLTH system for brain tumors and estimated the inhibition efficacy of tumor growth via an MRI-monitored long-term therapy in this study.

Long-term theranostic hydrogel system for solid tumors.

The long-term theranostic hydrogel system for solid tumors was prepared via simple physical mixing, which consisted of three major parts: the thermosensitive/biodegradable poly(organophosphazene) hydrogel, PEGylated cobalt ferrite nanoparticles, and paclitaxel (PTX). The PEGylated cobalt ferrite nanoparticles showed extremely low cytotoxicity due to the surface modification using PEG chains. The long-term theranostic hydrogel system showed adequate properties to be used for long-term MR theragnosis. In particular, the theranostic hydrogel gradually degraded over 28 days, and the PTX was sustainedly released out from the theranostic hydrogel over the same period in vitro. Furthermore, the in vivo efficacy of long-term MR theragnosis using the theranostic hydrogel system was estimated successfully over 3 weeks by using high field (4.7 T) animal MRI and solid tumor-bearing mice. Based on our results, we expect that this system can supply multiple data regarding a) the progress of therapy and b) the treatment processes via one- or two-time i.t. administration for cases in which surgical approaches are difficult to apply. Meanwhile, cancer patients can be free from the pain of multiple surgical treatments and have the advantage of therapy through a simple i.t. administration.

Thermosensitive/magnetic poly(organophosphazene) hydrogel as a long-term magnetic resonance contrast platform.

A thermosensitive/magnetic poly(organophosphazene) hydrogel (a magnetic hydrogel) was designed and synthesized for long-term magnetic resonance (MR) imaging. To turn a thermosensitive poly(organophosphazene) hydrogel (an original hydrogel) into a long-term MR contrast platform, cobalt ferrite (CoFe(2)O(4)) nanoparticles, which have hydrophobic surfaces, were bound to the original hydrogel via interactions between the hydrophobic surfaces of the nanoparticles and the (L)-isoleucine ethyl esters of the polymer. The magnetic hydrogel showed extremely low cytotoxicity and adequate magnetic properties for use in long-term MR imaging, in addition to possessing the same properties of the original hydrogel, such as viscosity, thermosensitivity, biodegradability, biocompatibility, a reversible sol-to-gel phase transition near body temperature, and injectability. The magnetic hydrogel was injected into a rat brain using stereotactic surgery. After the injection, the applicable potentiality as a long-term MR contrast platform was successfully estimated over 4-5 weeks. Consequently, it was shown that a magnetic hydrogel as a long-term MR contrast platform has the potential to be applied in a long-term theranostic hydrogel system. Furthermore, it is expected that this platform can be useful in the clinical field of incurable diseases due to either surgical difficulties or lethality, such as with brain tumors, when the platform is combined with therapeutic drugs for long-term MR theragnosis in further studies.

Design of polyphosphazene hydrogels with improved structural properties by use of star-shaped multithiol crosslinkers.

The feasibility of using a star-shaped crosslinker to produce a hydrogel with controlled mechanical properties and degradation rates was investigated. The aqueous blends of functional polymers and crosslinkers formed a solution at low temperature and a hydrogel with desired mechanical properties at body temperature. The introduction of star-shaped crosslinkers affected the microscopic and macroscopic properties of the hydrogel. The fabricated hydrogels could be suitable for many potential biomedical applications because of their injectability, tunable mechanical properties, controlled degradation rate and gel formation under physiological conditions.

The biological efficiency and bioavailability of human growth hormone delivered using injectable, ionic, thermosensitive poly(organophosphazene)-polyethylenimine conjugate hydrogels.

We have endeavored to develop injectable, thermosensitive, biodegradable hydrogels that prolong human growth hormone (hGH) release, improving bioavailability through introducing balanced ionic interactions. Cationic poly(organophosphazene)-polyethylenimine (PEI, 1.8 kDa) conjugate hydrogels were synthesized as those hydrogels for sustained delivery of anionic hGH with proper ionic strength of association/dissociation. We have additionally prepared different chain lengths of α-amino-ω-methoxy-poly(ethylene glycol) (AMPEG550 and AMPEG750) for the synthesis of conjugates as a means to control hydrogel degradation rates. All Aqueous solutions of PEI-conjugates became hydrogels hydrolyzable in proportion to AMPEG molecular weight at body temperature; these PEI-conjugates complexed with hGH and extended hGH release in vitro. In pharmacokinetic studies of hGH behavior in SD rats, hydrogels of PEI-conjugate/hGH complexes could suppress the initial burst-phase, and extend the duration, of release, as well as increasing of area under the curve (AUC) compared to controls including hGH solution or non-ionic hydrogel. In a hypophysectomized rat model, the biological efficacy of hGH delivered from PEI-conjugate/hGH complex hydrogels was equivalent to that from daily administration over four days based on body weight gain and width of the tibial growth plate. These results suggest that ionic, thermosensitive, poly(organophosphazene)-PEI-conjugate hydrogel demonstrates potential as an injectable depot for sustained delivery of bioavailable hGH.

Injectable, dual cross-linkable polyphosphazene blend hydrogels.

A new class of injectable, self cross-linkable, and thermosensitive polyphosphazene-based blending systems of functional thiolated and acrylated polymers was designed and synthesized to develop an ideal injectable carrier, and to overcome many barriers associated with developing the injectable carriers, such as the uses of monomeric crosslinkers, catalysts, oxidants, pH adjustments, initiators, UV light, heat production and organic solvent. The aqueous solutions of the polymer blends were exhibited a solution state at low temperature, and transformed into a hydrogel state with desired mechanical strength at body temperature via thermosensitive hydrophobic interactions. The mechanical strength was further improved by the cross-linking of thiol groups with acrylate groups in the polymer network under physiological conditions. The thermoresponsive hydrophobic interactions in the polymer network accelerated the chemical cross-linking to improve the mechanical property. The mechanical strength, inner three-dimensional network, and degradation rate can be tuned through the degree of cross-linking between the thermosensitive and functional blended polymers. The results suggest that the self cross-linkable thermosensitive polyphosphazene blend systems have great potentials to play a crucial role as an injectable carrier because of their improved suitable mechanical properties for application potentials, in addition to their inherent advantages such as injectable, biodegradable and thermosensitive properties.

Enhancement of sustained and controlled protein release using polyelectrolyte complex-loaded injectable and thermosensitive hydrogel.

In this study, we aimed at developing controlled and sustained protein release formulations using a combination system of polyelectrolyte complexes (PECs) and thermosensitive poly(organophosphazene) hydrogels as an injectable gel-depot system. In the protein-loaded hydrogel system, the loaded proteins were released rapidly through diffusion regardless of viscosities and mass loss of the gels because of the small hydrodynamic size of the proteins. To suppress protein diffusion and increase protein size, we induced PECs between negatively charged proteins (BSA, gelatin-type B 75 bloom, α-amylase, and hGH) and polycations (protamine, polyethylenimine, poly-l-lysine, and poly-l-arginine (PLA)) via an electrostatic interaction and loaded the PECs into the hydrogels. The formations of PECs were affected by molecular weight, pI (or pK(a)), and types of amine group of the used polycations. Unlike other polycations, PLA formed a large uniform complex with BSA, and the PLA/protein complex-loaded hydrogel showed the slowest protein release behavior. In the PEC-loaded hydrogel system, the protein release could also be controlled by viscosities of the gel and weight ratios of polycations and proteins, although the activities of the proteins were decreased in proportion to the PLA amounts. These results suggest that the PEC-loaded injectable and thermosensitive poly(organophosphazene) hydrogel has considerable potential for creating a sustained protein delivery system by using the PEC via electrostatic interaction.

Sustained delivery of human growth hormone using a polyelectrolyte complex-loaded thermosensitive polyphosphazene hydrogel.

A combined system of polyelectrolyte complex (PEC) and injectable, biodegradable and thermosensitive poly(organophosphazene) hydrogel has been suggested as an injectable depot for a controlled and sustained delivery of human growth hormone (hGH) to improve patient compliance. PEC was prepared by mixing polycations with hGH to suppress diffusion of hGH from the hydrogel through an enlargement of the hydrodynamic size of hGH. Among the polycations, poly-L-arginine (PLA) formed a large complex with hGH and its size increased as the amount of PLA increased. When PLA and/or zinc were added to hGH, the time-dependent stability of hGH increased more than that of native-hGH. The polymer solution containing PECs formed a gel at 37°C. PLA decreased the initial release rate of hGH in proportion to the amount of PLA in vitro and in vivo. Zinc increased the released amount of hGH from the PEC-loaded hydrogel in vitro and in vivo. In a pharmacokinetic study in rats, a single administration of PEC-loaded hydrogel resulted in the sustained release of hGH for 5days. These results suggest that injectable, biodegradable, and thermosensitive PEC-loaded poly(organophosphazene) hydrogel has great potential to be used as an effective delivery system for a sustained release of hGH with improved patient compliance.

Chemically crosslinkable thermosensitive polyphosphazene gels as injectable materials for biomedical applications.

Chemically crosslinkable and thermosensitive poly(organophosphazenes) containing multiple thiol (-SH) groups along with hydrophobic isoleucine ethyl ester and hydrophilic alpha-amino-omega-methoxy-poly(ethylene glycol) of the molecular weight 550 have been synthesized and characterized as an injectable biomaterial. The aqueous solutions of these polymers were transformed into hydrogel with desired gel strength at body temperature via hydrophobic interactions, and the gel strength was further improved by the cross-linking of thiol groups with crosslinkers, divinyl sulfone (VS) and PEG divinyl sulfone (PEGVS) under physiological conditions. The kinetics of cross-linking behavior of polymer thiol groups with crosslinkers was studied in both in vitro and in vivo conditions. Field Emission-Scanning Electron Microscopy (FE-SEM), swelling experiments, and rheology study of present polymers revealed that the inner three-dimensional hydrogel networks depended on the degree of thiol units in the polymer network. From the in vivo (in mice) degradation studies, the dual cross-linked gels showed to have a controlled degradation. These results demonstrate that the inner network of the hydrogels can be tuned, gel strength and degradation rate can be controlled, and the chemically crosslinkable and thermosensitive poly(organophosphazenes) hold promises for uses as injectable systems for biomedical applications including tissue engineering and protein delivery.

Thermosensitive poly(organophosphazene)-paclitaxel conjugate gels for antitumor applications.

A poly(organophosphazene)-PTX conjugate was synthesized by a covalent ester linkage between PTX and carboxylic acid-terminated poly(organophosphazene), which can be readily modified by various hydrophobic, hydrophilic, and other functional substitutes. The physicochemical properties, hydrolytic degradation and PTX release behaviors of the polymer-PTX conjugate were characterized, in addition to the in vitro and in vivo antitumor activities. The aqueous solutions of these conjugates showed a sol-gel transition behavior that depended on temperature changes. The in vitro antitumor activity of the polymer-PTX conjugate was investigated by an MTT assay against human tumor cell lines. From the in vivo antitumor activity studies with tumor-induced (xenografted) nude mice, the polymer-paclitaxel conjugate hydrogels after local injection at the tumor site were shown to inhibit tumor growth more effectively and longer than paclitaxel and saline alone, indicating that the tumor-active paclitaxel from the polymer-PTX conjugate hydrogel is released slowly over a longer period of time and effectively accumulated locally in the tumor sites. These combined observations suggest that this poly(organophosphazene)-PTX conjugate holds promise for use in clinical studies as single and/or combination therapies.

The use of injectable, thermosensitive poly(organophosphazene)-RGD conjugates for the enhancement of mesenchymal stem cell osteogenic differentiation.

An injectable and thermosensitive poly(organophosphazene)-RGD conjugate to enhance functionality was synthesized by a covalent amide linkage between a cell adhesion peptide, GRGDS and carboxylic acid-terminated poly(organophosphazene). The aqueous solutions of synthesized poly(organophosphazene)-GRGDS conjugates existed in an injectable fluid state at room temperature and immediately formed a hydrogel at body temperature. The rabbit mesenchymal stem cells (rMSCs) on the polymer-GRGDS conjugate (conjugate 1-2, 0.05 mol fraction as GRGDS) hydrogel constructs using an injection method into a nude mouse were proved to express markers at mRNA level for all stages towards osteogenesis and mainly a sharp increase of osteocalcin (OCN, a typical late osteogenic differentiation marker) levels at 4th week post-induction indicated that the maturation process has started within this period. By histological and immunohistochemical evaluations, significantly high mineralization level by calcium contents was detected qualitatively and collagen type I (Col I), a major characteristic marker protein, was mainly and highly expressed by the rMSCs cultivated in the polymer-GRGDS conjugate hydrogel constructs formed into the nude mouse. The results suggest that the poly(organophosphazene)-GRGDS conjugate to enhance biofunctionality hold a promise for cell delivery material to induce osteogenic differentiation of MSC for enhancing ectopic bone formation.

Doxorubicin-polyphosphazene conjugate hydrogels for locally controlled delivery of cancer therapeutics.

Poly(organophosphazene)-doxorubicin (DOX) conjugate bearing hydrophobic L-isoleucine ethyl ester (IleOEt) and hydrophilic alpha-amino-omega-methoxy-poly(ethylene glycol) with molecular weight of 550 Da (AMPEG 550) along with carboxylic acid as a functional group was synthesized to create a drug delivery system, which is based on locally injectable, biodegradable, and thermosensitive hydrogels. In addition to the evaluation of the in vitro and in vivo antitumor activities, the physicochemical properties, hydrolytic degradation, and DOX release profile of the poly(organophosphazene)-DOX conjugate were determined. The aqueous solution of the polymer-DOX conjugate showed a sol-gel transition behavior depending on temperature changes. Based on the in vivo antitumor activities of the locally injected poly(organophosphazene)-DOX conjugate into the tumor-induced nude mice, the conjugate hydrogel after the local injection at the tumor site was shown to inhibit tumor growth more effectively with less toxicity and much longer than doxorubicin and saline as controls, indicating that tumor active DOX from the conjugate hydrogel is released slowly over a longer period of time and effectively accumulated locally in the tumor sites. These results suggest that the poly(organophosphazene)-doxorubicin conjugates hold great potential for use in preclinical and clinical studies as single and/or combination therapies.