Thermoresponsive and Biodegradable Amphiphilic Block Copolymers with Pendant Functional Groups.

BACKGROUND: To develop the biodegradability and thermoresponsive hydrogel, in this work we designed a pendant-functionalized, thermoresponsive, amphiphilic block copolymer. METHODS: Methoxy poly(ethylene glycol) (MPEG)-b-[poly(ε-caprolactone)-ran-poly(ε-caprolactone-3-one)-ran-polylactic acid] (MCL) and (MPEG-b-[PCL-ran-POD-ran-PLA]) [MCL-(CO)] block copolymers were prepared by ring-opening polymerization of ε-caprolactone, OD and lactide monomers. The subsequent derivatization of MCL-(CO) provided MPEG-b-[PCL-ran-poly(ε-caprolactone-3-COOH)-ran-PLA] [MCL-(COOH)] with COOH pendant groups and MPEG-b-[PCL-ran-poly(ε-caprolactone-3-NH2)-ran-PLA] [MCL-(NH2)] with NH2 pendant groups. RESULTS: The measured segment ratios of MCL-(CO), MCL-(COOH), and MCL-(NH2) agreed well with the target ratios. The abundances of the COOH and NH2 groups in the MCL-(COOH) and MCL-(NH2) copolymers were determined by 1H- and 13C-nuclear magnetic resonance spectroscopy, and agreed well with the target abundances. MCL-(CO), MCL-(COOH), and MCL-(NH2) formed homogeneous, white, opaque emulsions at room temperature. Rheological analysis of the block copolymer suspensions indicated a solution-to-hydrogel phase transition as a function of temperature. The solution-to-hydrogel phase transitions and the biodegradation of MCL-(CO), MCL-(COOH), and MCL-(NH2) were affected by varying the type (ketone, COOH, or NH2) and abundance of the pendant groups. CONCLUSION: MCL-(CO), MCL-(COOH), and MCL-(NH2) with ketone, COOH, and NH2 pendant groups showed solution-to-hydrogel phase transitions and biodegradation behaviors that depended on both the type and number of pendant groups.

An injectable, electrostatically interacting drug depot for the treatment of rheumatoid arthritis.

To the best of our knowledge, no studies have yet examined the electrostatic interaction of polyelectrolytes with electrolyte drugs for the treatment of rheumatoid arthritis (RA). Here, an injectable, electrostatically interacting drug depot is described. We prepared methoxy polyethylene glycol-b-poly(ε-caprolactone)-ran-poly(l-lactic acid) (MC) diblock copolymers with a carboxylic acid group (MC-C) at the pendant position. MC-C was polyelectrolytes that exhibited negative zeta potentials. Sulfasalazine [Sul(-)] and minocycline [Min(+)], electrolyte RA drugs, exhibited negative and positive zeta potentials, respectively. The electrolyte RA drugs were loaded into the polyelectrolyte MC-C solution to prepare injectable, electrostatically interacting depot formulations. The formulation with an attractive electrostatic interaction [Min(+)-MC-C] exhibited gradual release of Min(+) from the MC-C depot over an extended period and suppressed the growth of inflammatory RAW 264.7 cells without affecting synovial cells. Mature chondrocytes were observed after H&E and safranin O staining of the cartilage of Min(+)-MC-C intra-articularly injected RA-induced rats. In comparison with other formulations, Min(+)-MC-C induced the suppression of the expression of pro-inflammatory proteins TNF-α and IL-1ß in the articular knee joint, which resulted in the amelioration of RA. In conclusion, an injectable, electrostatically interacting depot formulation administered through intra-articular injection successfully provided almost complete amelioration of RA.

BMP2-modified injectable hydrogel for osteogenic differentiation of human periodontal ligament stem cells.

This is the first report on the development of a covalently bone morphogenetic protein-2 (BMP2)-immobilized hydrogel that is suitable for osteogenic differentiation of human periodontal ligament stem cells (hPLSCs). O-propargyl-tyrosine (OpgY) was site-specifically incorporated into BMP2 to prepare BMP2-OpgY with an alkyne group. The engineered BMP2-OpgY exhibited osteogenic characteristics after in vitro osteogenic differentiation of hPLSCs, indicating the osteogenic ability of BMP2-OpgY. A methoxy polyethylene glycol-(polycaprolactone-(N3)) block copolymer (MC-N3) was prepared as an injectable in situ-forming hydrogel. BMP2 covalently immobilized on an MC hydrogel (MC-BMP2) was prepared quantitatively by a simple biorthogonal reaction between alkyne groups on BMP2-OpgY and azide groups on MC-N3 via a Cu(I)-catalyzed click reaction. The hPLSCs-loaded MC-BMP2 formed a hydrogel almost immediately upon injection into animals. In vivo osteogenic differentiation of hPLSCs in the MC-BMP2 formulation was confirmed by histological staining and gene expression analyses. Histological staining of hPLSC-loaded MC-BMP2 implants showed evidence of mineralized calcium deposits, whereas hPLSC-loaded MC-Cl or BMP2-OpgY mixed with MC-Cl, implants showed no mineral deposits. Additionally, MC-BMP2 induced higher levels of osteogenic gene expression in hPLSCs than in other groups. In conclusion, BMP2-OpgY covalently immobilized on MC-BMP2 induced osteogenic differentiation of hPLSCs as a noninvasive method for bone tissue engineering.

Preparation of Biodegradable and Elastic Poly(ε-caprolactone-co-lactide) Copolymers and Evaluation as a Localized and Sustained Drug Delivery Carrier.

To develop a biodegradable polymer possessing elasticity and flexibility, we synthesized MPEG-b-(PCL-co-PLA) copolymers (PCxLyA), which display specific rates of flexibility and elasticity. We synthesize the PCxLyA copolymers by ring-opening polymerization of ε-caprolactone and l-lactide. PCxLyA copolymers of various compositions were synthesized with 500,000 molecular weight. The PCxLyA copolymers mechanical properties were dependent on the mole ratio of the ε-caprolactone and l-lactide components. Cyclic tensile tests were carried out to investigate the resistance to creep of PCxLyA specimens after up to 20 deformation cycles to 50% elongation. After in vivo implantation, the PCxLyA implants exhibited biocompatibility, and gradually biodegraded over an eight-week experimental period. Immunohistochemical characterization showed that the PCxLyA implants provoked in vivo inflammation, which gradually decreased over time. The copolymer was used as a drug carrier for locally implantable drugs, the hydrophobic drug dexamethasone (Dex), and the water-soluble drug dexamethasone 21-phosphate disodium salt (Dex(p)). We monitored drug-loaded PCxLyA films for in vitro and in vivo drug release over 40 days and observed real-time sustained release of near-infrared (NIR) fluorescence over an extended period from hydrophobic IR-780- and hydrophilic IR-783-loaded PCxLyA implanted in live animals. Finally, we confirmed that PCxLyA films are usable as biodegradable, elastic drug carriers.

Preparation of Pendant Group-Functionalized Diblock Copolymers with Adjustable Thermogelling Behavior.

Recently, several thermogelling materials have been developed for biomedical applications. In this study, we prepared methoxy polyethylene glycol (MPEG)-b-(poly(ε-caprolactone)-ran-poly(2-chloride-ε-caprolactone) (PCL-ran-PfCL)) (MP-Cl) diblock copolymers at room temperature via the ring-opening polymerization of caprolactone (CL) and 2-chloride-ε-caprolactone (fCL) monomers, using the terminal alcohol of MPEG as the initiator in the presence of HCl. MPEG-b-(poly(ε-caprolactone)-ran-poly(2-azide-ε-caprolactone) (PCL-ran-PCL-N3)) (MP-N3) was prepared by the reaction of MP-Cl with sodium azide. MPEG-b-(poly(ε-caprolactone)-ran-poly(2-amine-ε-caprolactone) (PCL-ran-PCL-NH2)) (MP-NH2) was subsequently prepared by Staudinger reaction. MP-Cl and MP-N3 showed negative zeta potentials, but MP-NH2 had a positive zeta potential. MP-Cl, MP-N3, and MP-NH2 solutions formed opaque emulsions at room temperature. The solutions exhibited a solution-to-hydrogel phase transition as a function of the temperature and were affected by variation of the chloride, azide, and the amine pendant group, as well as the amount of pendant groups present in their structure. Additionally, the phase transition of MP-Cl, MP-N3, and MP-NH2 copolymers was altered by pendant groups. The solution-to-hydrogel phase transition was adjusted by tailoring the crystallinity and hydrophobicity of the copolymers in aqueous solutions. Collectively, MP-Cl, MP-N3, and MP-NH2 with various pendant-group contents in the PCL segment showed a solution-to-hydrogel phase transition that depended on both the type of pendant groups and their content.

In Vivo Osteogenic Differentiation of Human Dental Pulp Stem Cells Embedded in an Injectable In Vivo-Forming Hydrogel.

In this study, human dental pulp stem cells (hDPSCs) are examined as a cellular source for bone tissue engineering using an in vivo-forming hydrogel. The hDPSCs are easily harvested in large quantities from extracted teeth. The stemness of harvested hDPSCs indicates their relative tolerance to ex vivo manipulation in culture. The in vitro osteogenic differentiation of hDPSCs is characterized using Alizarin Red S (ARS), von Kossa (VK), and alkaline phosphatase (ALP) staining. The solution of hDPSCs and a methoxy polyethylene glycol-polycaprolactone block copolymer (PC) is easily prepared by simple mixing at room temperature and in no more than 10 s it forms in vivo hydrogels after subcutaneous injection into rats. In vivo osteogenic differentiation of hDPSCs in the in vivo-forming hydrogel is confirmed by micro-computed tomography (CT), histological staining, and gene expression. Micro-CT analysis shows evidence of significant tissue-engineered bone formation in hDPSCs-loaded hydrogel in the presence of osteogenic factors. Differentiated osteoblasts in in vivo-forming hydrogel are identified by ARS and VK staining and are found to exhibit characteristic expression of genes like osteonectin, osteopontin, and osteocalcin. In conclusion, hDPSCs embedded in an in vivo-forming hydrogel may provide benefits as a noninvasive formulation for bone tissue engineering applications.

Preparation and investigation of hydrolyzed polyacrylonitrile as a preliminary biomedical hydrogel.

BACKGROUND: Hydrolyzed polyacrylonitrile (HPAN) has attracted much attention as a hydrogel for a broad range of biomedical applications. Therefore, in this study, we prepared HPAN derivatives with controllable compositions by the radical polymerization of acrylonitrile (AN), methacrylic acid (MAA) and N-isopropylacrylamide (NIPAM) monomers. RESULTS: The prepared poly(AN-co-MAA-co-NIPAM) copolymers had different ratios of AN, MAA, and NIPAM and molecular weights ranging from 2000 to 50,000. The copolymers were prepared as films to examine their properties. The prepared copolymer films showed different solubilities, contact angles, and swelling ratios. The properties of the copolymer films were affected by the hydrophobic PAN segments and the hydrophilic PMAA or PNIPAM segments. CONCLUSION: Thus, we conclude that introducing PMAA and PNIPAM segments with different ratios and lengths into PAN segments could represent a method of controlling the hydrogel properties of copolymers.

Biodegradable poly(lactide-co-glycolide-co-ε-caprolactone) block copolymers - evaluation as drug carriers for a localized and sustained delivery system.

To develop an appropriate drug carrier for drug delivery systems, we prepared random poly(lactide-co-glycolide-co-ε-caprolactone) (PLGC) copolymers in comparison to commercial poly(lactic acid-co-glycolic acid) (PLGA) grades. The molecular weights of PLGC copolymers varied from 20k to 90k g mol-1 in the total polyester segments, when poly-l-lactic acid (PLLA), polyglycolic acid (PGA), and polycaprolactone (PCL) compositions were kept constant. The lengths of PLGC copolymers varied from 10 : 10 : 80 to 40 : 40 : 20 in the PLLA : PGA : PCL segments, when the molecular weights of the total polyester segments were kept constant. The crystalline properties of the PLGA copolymers can be changed to amorphous by the incorporation of PCL segments. In vitro and in vivo degradation behavior can be easily tuned from a few days to a few weeks by changing the chemical composition of the PLGC copolymers. The in vivo inflammation associated with the PLGC implants was less pronounced than that associated with PLGA. In this study, as drug delivery carriers for locally implantable paclitaxel (Ptx) dosages, Ptx-loaded PLGC and PLGA films showed in vitro and in vivo Ptx release for 35 days. The orders of Ptx release showed profiles similar to those of in vitro and in vivo degradation of PLGC. Using near-infrared (NIR) fluorescence imaging, we confirmed the sustained release of NIR over an extended period from IR-780-loaded PLGC and PLGA implanted in live animals. In conclusion, we confirmed that compared to PLGA, PLGC effectively acts as a drug carrier for drug delivery systems.

Chfr is linked to tumour metastasis through the downregulation of HDAC1.

Chfr is a ubiquitin ligase that functions in the mitotic checkpoint by delaying entry into metaphase in response to mitotic stress. It has been suggested that Chfr is a tumour suppressor as Chfr is frequently silenced in human cancers. To better understand how Chfr activity relates to cell-cycle progression and tumorigenesis, we sought to identify Chfr-interacting proteins using affinity purification combined with mass spectrometry. Histone deacetylase 1 (HDAC1), which represses transcription by deacetylating histones, was newly isolated as a Chfr-interacting protein. Chfr binds and downregulates HDAC1 by inducing its polyubiquitylation, both in vitro and in vivo. Ectopic expression of Chfr in cancer cells that normally do not express it results in downregulation of HDAC1, leading to upregulation of the Cdk inhibitor p21(CIP1/WAF1) and the metastasis suppressors KAI1 and E-cadherin. Coincident with these changes, cells arrest in the G1 phase of the cell cycle and become less invasive. Collectively, our data suggest that Chfr functions as a tumour suppressor by regulating HDAC1.