Substantial Confinement of Crystal Growth of Organic Crystalline Materials in Metal-Organic Membrane Microshells.

This study proposes a robust microshell encapsulation system in which a metal-organic membrane (MOM), consisting of phytic acids (PAs) and metal ions, intrinsically prevents the molecular crystal growth of organic crystalline materials (OCMs). To develop this system, OCM-containing oil-in-water (O/W) Pickering emulsions were enveloped with the MOM, in which anionic pulp cellulose nanofiber (PCNF) primers electrostatically captured zinc ions at the O/W interface and chelated with PA, thus producing the MOM with a controlled shell thickness at the micron scale. We ascertained that the MOM formation fills and covers ∼75% of the surface pore size of PCNF films, which enhances the interfacial modulus by 2 orders of magnitude compared to that when treated with bare PCNFs. Through a feasibility test using a series of common OCMs, including ethylhexyl triazone, avobenzone, and ceramide, we demonstrated the excellent ability of our MOM microshell system to stably encapsulate OCMs while retaining their original molecular structures over time. These findings indicate that our MOM-reinforced microshell technology can be applied as a platform to substantially confine the crystal growth of various types of OCMs.

Effects of pH and buffer concentration on the thermal stability of etanercept using DSC and DLS.

The protein size, electrical interaction, and conformational stability of etanercept (marketed as Enbrel®) were examined by thermodynamic and light scattering methods with changing pH and buffer concentration. As pH of etanercept increased from pH 6.6 to 8.6, electrical repulsion in the solution increased, inducing a decrease in protein size. However, the size changed less in high buffer concentration and irreversible aggregation issues were not observed; in contrast, aggregates of about 1000 nm were observed in low buffer concentration at the pH range. Three significant unfolding transitions (Tm) were observed by differential scanning calorimetry (DSC). Unlikely to Tm1, Tm2 and Tm3 were increased as the pH increased. Higher Tm at high buffer concentration was observed, indicating increased conformational stability. The apparent activation energy of unfolding was further investigated since continuous increase of Tm2 and Tm3 was not sufficient to determine optimal conditions. A higher energy barrier was calculated at Tm2 than at Tm3. In addition, the energy barriers were the highest at pH from 7.4 to 7.8 where higher Tm1 was also observed. Therefore, the conformational stability of protein solution significantly changed with pH dependent steric repulsion of neighboring protein molecules. An optimized pH range was obtained that satisfied the stability of all three domains. Electrostatic circumstances and structural interactions resulted in irreversible aggregation at low buffer concentrations and were suppressed by increasing the concentration. Therefore, increased buffer concentration is recommended during protein formulation development, even in the earlier stages of investigation, to avoid protein instability issues.

Injectable in situ-forming pH/thermo-sensitive hydrogel for bone tissue engineering.

We developed a novel pH- and thermo-sensitive hydrogel as a scaffold for autologous bone tissue engineering. We synthesized this polymer by adding pH-sensitive sulfamethazine oligomers (SMOs) to both ends of a thermo-sensitive poly(epsilon-caprolactone-co-lactide)-poly(ethylene glycol)-poly(epsilon-caprolactone-co-lactide) (PCLA-PEG-PCLA) block copolymer, yielding a pH/thermo-sensitive SMO-PCLA-PEG-PCLA-SMO block copolymer. The synthesized block copolymer solution rapidly formed a stable gel under physiological conditions (pH 7.4 and 37 degrees C), whereas it formed a sol at pH 8.0 and 37 degrees C, making it injectable. This pH/thermo-sensitive hydrogel exhibited high biocompatibility in a Dulbecco's modified Eagle's medium extract test. Under physiological conditions, the hydrogel easily encapsulated human mesenchymal stem cells (hMSCs) and recombinant human bone morphogenetic protein-2 (rhBMP-2), with encapsulating efficiencies of about 90% and 85%, respectively. To assay for ectopic bone formation in vivo, we subcutaneously injected a polymer solution containing hMSCs and rhBMP-2 into the back of mice, after which we could observe hMSC differentiation for up to 7 weeks. Histological studies revealed mineralized tissue formation and high levels of alkaline phosphatase activity in the mineralized tissue. Therefore, this pH/thermo-sensitive SMO-PCLA-PEG-PCLA-SMO block copolymer demonstrated potential as an injectable scaffold for bone tissue engineering, with in situ formation capabilities.

pH- and temperature-sensitive, injectable, biodegradable block copolymer hydrogels as carriers for paclitaxel.

Paclitaxel (PTX) was loaded into synthetic pH/T-sensitive block copolymer (OSM-PCLA-PEG-PCLA-OSM) solution with various concentrations. The phase diagram of PTX-loaded block copolymer solution shifted to lower temperature region compared to net block copolymer because of the salting-out effect of PTX. Release profiles of PTX showed sustained manner regardless of loading amount of PTX. To evaluate anti-tumor effect of PTX-loaded block copolymer, solutions were injected subcutaneously to tumor-bearing mice and TUNEL assay examined. PTX-loaded block copolymer hydrogel for in vivo use showed good anti-tumor effect for 2 weeks and induced strong apoptosis in tumor tissue. Therefore, we conclude OSM-PCLA-PEG-PCLA-OSM block copolymer as an effective injectable carrier of PTX.

Sulfonamide-based pH- and temperature-sensitive biodegradable block copolymer hydrogels.

Novel pH- and temperature-sensitive biodegradable poly(epsilon-caprolactone-co-lactide)-poly(ethylene glycol) (PCLA-PEG) block copolymers were synthesized with oligomeric sulfamethazine (OSM) end groups (OSM-PCLA-PEG-PCLA-OSM). Aqueous solutions of these block copolymers have shown sol-gel transition behavior upon both temperature and pH changes under physiological conditions (37 degrees C, pH 7.4). The sol-gel transition of these block copolymer solutions was fine-tuned by controlling the PEG length, the hydrophobic to hydrophilic block ratio (PCLA/PEG), and the molecular weight of the sulfamethazine oligomer. Since changes in temperature do not induce gel formation in this pH- and temperature-sensitive block copolymer solution, this hydrogel can be employed as an injectable carrier using a long guide catheter into the body. In addition, the pH of the block copolymer solution showed no change following PCLA degradation over 1 month, and no indication of gel collapse was observed on addition of buffer solution. As such, these properties make the OSM-PCLA-PEG-PCLA-OSM hydrogel an ideal candidate for use as an injectable carrier for certain protein-based drugs known to denature in low-pH environments.

Biodegradability and biocompatibility of a pH- and thermo-sensitive hydrogel formed from a sulfonamide-modified poly(epsilon-caprolactone-co-lactide)-poly(ethylene glycol)-poly(epsilon-caprolactone-co-lactide) block copolymer.

A pH- and thermo-sensitive block copolymer was synthesized by adding pH-sensitive sulfamethazine oligomers (SMOs) to either end of a thermo-sensitive poly(epsilon-caprolactone-co-lactide)-poly(ethylene glycol)-poly(epsilon-caprolactone-co-lactide) (PCLA-PEG-PCLA) block copolymer. The resulting pH- and thermo-sensitive SMO-PCLA-PEG-PCLA-SMO block copolymer solution did not form a gel at high pH (pH 8.0) or at increased temperatures (ca. 70 degrees C), but did form a stable gel under physiological conditions (pH 7.4 and 37 degrees C). The degradation rate of the pH- and thermo-sensitive block copolymer decreased substantially compared with the control block copolymer of PCLA-PEG-PCLA, due to the buffering effect of the SMO-PCLA-PEG-PCLA-SMO sulfonamide groups on the acidic monomer-induced rapid degradation of PCLA-PEG-PCLA. This suitable sol-gel transition and sustained biodegradability of the pH- and thermo-sensitive SMO-PCLA-PEG-PCLA-SMO block copolymer resolves two of the major drawbacks associated with thermo-sensitive block copolymers, namely premature gelation and rapid degradation. Interestingly, SMO-PCLA-PEG-PCLA-SMO showed no evidence of cytotoxicity in vitro. However, subcutaneous injection of the pH- and thermo-sensitive block copolymer solution (20wt% in PBS at pH 8.0) into Sprague-Dawley (SD) rats resulted in rapid, stable gel formation, with the injected hydrogel being completely degraded in vivo in just 6 weeks. The injected hydrogel in vivo presented a typical acute inflammation within 2 weeks, although chronic inflammation was not observed during the first 6-week period. As such, the pH- and thermo-sensitive hydrogel of the SMO-PCLA-PEG-PCLA-SMO block copolymer is a suitable candidate for use in drug delivery systems and cell therapy.

Novel pH sensitive block copolymer micelles for solvent free drug loading.

Novel pH sensitive biodegradable block copolymers (MPEG-PDLLA-OSM) composed of mono-methoxy poly(ethylene glycol) (MPEG), poly (D,L-lactide) (PDLLA) and sulfamethazine oligomer (OSM) were synthesized via ring-opening polymerization and a dicyclohexyl carboimide (DCC) coupling reaction. These copolymers had a relatively low critical micelle concentration (CMC) due to the strong hydrophobic properties of non-ionized OSM at pH 7.0. Also, the pH sensitive block copolymers showed the micelle-unimer transition due to the ionization-non-ionization of OSM in the pH range (pH 7.2-8.4) above the CMC. Due to the pH sensitive properties of the block copolymer, the hydrophobic drug paclitaxel (PTX) was incorporated into a pH sensitive block copolymer micelle by the pH induced micellization method, without using an organic solvent. The block copolymer micelle prepared by pH induced micellization showed a relatively high PTX loading efficiency, and good stability for 2 d at 37 degrees C. Furthermore, the PTX loaded micelle showed a sustained release of PTX with a small burst in vitro over 2 d. The present results suggest that the pH induced micellization method due to the micelle-unimer transition of the pH sensitive block copolymer would be a novel and valuable drug incorporation tool for hydrophobic and protein drugs, since no organic solvent is involved in the formulation.

Novel injectable pH and temperature sensitive block copolymer hydrogel.

A novel pH and temperature sensitive block copolymer was prepared by adding pH sensitive moiety to temperature sensitive block copolymer. This block copolymer solution showed a reversible sol-gel transition by a small pH change in the range of pH 7.4-8.0 and also by the temperature change in the region of body temperature. The very precise molecular weight control of block copolymer and the prudential tuning of hydrophilic-hydrophobic balance were needed to control the phase diagram. This block copolymer solution forms a gel at 37 degrees C, pH 7.4 (human body). When the block copolymer solution is at room temperature and pH 8.0 as a sol state, both the temperature and pH change are needed for the gelation. This material can be employed as injectable carriers for hydrophobic drugs and proteins, etc. Gelation inside the needle can be prevented by an increase in the temperature during injection, because it does not change into the gel form with only increasing temperature. This material can be used for even a long guide catheter into the body. The block copolymer hydrogel which shows the sol-gel transition by the small pH change from pH 8.0 to pH 7.4 has merits in the delivery system for protein and cells which show cytotoxicity in acidic (below pH 6.5) or basic (above pH 8.5) conditions. This block copolymer system could be used as a template technology for injectable delivery systems.

Poly(D,L-lactic acid-co-glycolic acid)-b-poly(ethylene glycol)-b-poly (D,L-lactic acid-co-glycolic acid) triblock copolymer and thermoreversible phase transition in water.

Novel thermoreversible gelation behavior of aqueous solutions of ABA-type triblock copolymers composed of the central polyethylene oxide (PEG) block and two poly(D,L-lactic acid-co-glycolic acid) side blocks was found. Phase transition characteristics, such as critical gel concentration (CGC) and lower and upper critical gel temperature (CGT), are closely related to the molecular structure of the triblock copolymers. The CGC and the lower CGT both increases with increasing PEG/PLGA molecular weight ratio. Increasing the GA content in PLGA block induces a somewhat higher CGC. The copolymer forms micelles with a PLGA loop core and a PEG shell in water. Also grouped micelles are identified seemingly due to the bridging of two micelles sharing two PLGA blocks of a block copolymer chain. As the temperature increases the association of micelles increases, which results in gelation. The ABA-type copolymers exhibit a relatively low CGC (<10%) and low sol-gel transition temperatures compared to BAB-type copolymers. As the temperature increases further gel-sol transition is observed, which would result from the shrinkage of micelles with temperature increase. The hydrodynamic size of the micelles is monitored by dynamic laser scattering, and a possible gelation mechanism was suggested.