Adjustable bioadhesive control of PEGylated hyperbranch brushes on polystyrene microplate interface for the improved sensitivity of human blood typing.

A PEGylated 96-well polystyrene (PS) microplate was first introduced for applications in high-throughput screening for selective blood typing to minimize the risks in blood transfusions. Herein, we present a hemocompatible PS 96-well microplate with adjustable PEGylated hyperbranch brush coverage prepared by ozone pretreated activation and thermally induced surface PEGylation. The grafting properties, hydration capacity, and blood compatibility of the PEGylated hyperbrush immobilized PS surfaces in human blood were illustrated by the combined chemical and physical properties of the surface, and the dependence of the specific absorption of human plasma fibrinogen onto the PEGylated surfaces on the grafting density was analyzed by monoclonal antibodies. The surface coverage of PEGylated brushes plays a major role in the bioadhesive properties of modified PS microplates, which in turn control the level of agglutination sensitivity in blood typing. The bioadhesive resistance toward proteins, platelets, and erythrocytes in human whole blood showed a correlation to the controlled hydration properties of the PEGylated hyperbrush-modified surfaces. Therefore, we suggested that the surface coverage of PEGylated hyperbrushes on PS surfaces can increase the sensitivity of cross-matching blood agglutination by up to 16-fold compared to that of the conventional 96-well virgin PS due to the regulated biorecognition of hematocrit and antibodies of the PEGylated hyperbrush-modified surfaces.

Bioadhesive control of plasma proteins and blood cells from umbilical cord blood onto the interface grafted with zwitterionic polymer brushes.

In this work, bioadhesive behavior of plasma proteins and blood cells from umbilical cord blood (UCB) onto zwitterionic poly(sulfobetaine methacrylate) (polySBMA) polymer brushes was studied. The surface coverage of polySBMA brushes on a hydrophobic polystyrene (PS) well plate with surface grafting weights ranging from 0.02 mg/cm(2) to 0.69 mg/cm(2) can be effectively controlled using the ozone pretreatment and thermal-induced radical graft-polymerization. The chemical composition, grafting structure, surface hydrophilicity, and hydration capability of prepared polySBMA brushes were determined to illustrate the correlations between grafting properties and blood compatibility of zwitterionic-grafted surfaces in contact with human UCB. The protein adsorption of fibrinogen in single-protein solutions and at complex medium of 100% UCB plasma onto different polySBMA brushes with different grafting coverage was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The grafting density of the zwitterionic brushes greatly affects the PS surface, thus controlling the adsorption of fibrinogen, the adhesion of platelets, and the preservation of hematopoietic stem and progenitor cells (HSPCs) in UCB. The results showed that PS surfaces grafted with polySBMA brushes possess controllable hydration properties through the binding of water molecules, regulating the bioadhesive and bioinert characteristics of plasma proteins and blood platelets in UCB. Interestingly, it was found that the polySBMA brushes with an optimized grafting weight of approximately 0.1 mg/cm(2) at physiologic temperatures show significant hydrated chain flexibility and balanced hydrophilicity to provide the best preservation capacity for HSPCs stored in 100% UCB solution for 2 weeks. This work suggests that, through controlling grafting structures, the hemocompatible nature of grafted zwitterionic polymer brushes makes them well suited to the molecular design of regulated bioadhesive interfaces for use in the preservation of HSPCs from human UCB.

Extended release of bevacizumab by thermosensitive biodegradable and biocompatible hydrogel.

The antibody bevacizumab (Avastin) has been used clinically to treat intraocular neovascular diseases based on its antivascular endothelial growth factor (VEGF) character. The anti-VEGF strategy for retinal neovascular diseases is limited by the short half-life of bevacizumab and thus requires frequent injections. This Article reports the sustained release of bevacizumab from a biocompatible material that is composed of a triblock copolymer of poly(2-ethyl-2-oxazoline)-b-poly(ε-caprolactone)-b-poly(2-ethyl-2-oxazoline) (PEOz-PCL-PEOz). The amphiphilic PEOz-PCL-PEOz triblock copolymer was synthesized in three steps. First, the PEOz was polymerized by methyl p-toluenesulfonate and 2-ethyl-2-oxazoline (EOz), and the living end was terminated by potassium hydroxide methanolic solution. Subsequently, the hydroxyl-PEOz was used as a macroinitiator for the ring-opening polymerization of ε-caprolactone using a Tin(II) octoate catalyst to synthesize the telechelic hydroxylated PEOz-PCL. Finally, the PEOz-PCL-PEOz triblock copolymer was obtained using the 1,6-hexamethylene diisocyanateas a coupling reagent. The PEOz-PCL-PEOz was chemically and molecularly characterized by GPC, (1)H NMR, and FTIR, and its aqueous solution (ECE hydrogel) showed a reversible sol (room temperature)-gel (physiological temperature) phase transition, which serves as an easy antibody-packing system with extended release. The biodegradability of ECE hydrogel was assessed by the porosity formation at different periods by scanning electron microscopy. The ECE hydrogel had no in vitro cytotoxicity on the human retinal pigment epithelial cell line by flow cytometry. The histomorphology and electrophysiology of the rabbit neuroretina were preserved after 2 months of intravitreal injection. In conclusion, the ECE hydrogel has a temperature-sensitive sol-gel phase transition and is effective in vitro. Its intraocular biocompatibility demonstrated its great potential to be widely used in biomedical applications for extended drug release.

Sterically polymer-based liposomal complexes with dual-shell structure for enhancing the siRNA delivery.

The sterically polymer-based liposomal complexes (SPLexes) were formed by cationic polymeric liposomes and pH-sensitive diblock copolymer were studied for their capabilities in improving the stability with high efficiency of siRNA delivery. The SPLexes were formed a dual-shelled structure and uniform size distribution. The PEGylated outer shell could mitigate the phagocytosis and reduce the cytotoxicity. Moreover, the folated SPLexes improved 42.9× accumulation in vitro and 1.7× tumor uptake in vivo in contrast with nonfolated SPLexes. The protonated copolymer at low pH would improve the siRNA released into cytoplasm following SPLexes fusion with the endo/lysosome membrane and inhibited the protein expression to 75.6 ± 4.5% efficiently. Results of this study significantly contribute to efforts to develop lipoplexes based siRNA delivery systems.

In vitro evaluation of hexagonal polymeric micelles in macrophage phagocytosis.

This paper develops a non-spherical polymeric micelle using an amphiphilic block copolymer and a porphyrin crystalline structure. The nanoscale polymer micelles were characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM), revealing particle sizes of approximately 150 nm with a particular shape in the hexagonal lattice. The shape shows the selective uptake efficacy for the HeLa and macrophage cells, and inhibits phagocytosis against the macrophage.

Doxorubicin-Loaded Mixed Micelles Using Degradable Graft and Diblock Copolymers to Enhance Anticancer Sensitivity.

In this study, a graft copolymer, poly(N-(2-hydroxypropyl) methacrylamide dilactate)-co-(N-(2-hydroxypropyl) methacrylamide-co-histidine)-graft-poly(d,l-lactide), and a diblock copolymer, methoxy poly(ethylene glycol)-b-poly(d,l-lactide), were assembled into a mixed micellar system to encapsulate the anticancer drug doxorubicin (Dox). This mixed micellar system possesses the hydrophobic lactide segment of both copolymers, which reinforces its stability in physiological milieus; the histidine molecules appended on the graft copolymer provide the desired pH-responsive behavior to release Dox during internalization in cancer cells. The results demonstrate that the two copolymers were successfully prepared, and their ratios in the mixed micelles were optimized on the basis of the results of the stability tests. Under acidic conditions, the mixed micelles swell and are able to release their payloads. Therefore, the in vitro results indicate that the Dox in the mixed micelles is released effectively in response to the environmental pH of the mimetic internalization process, increasing cancer cells' sensitivity toward Dox. The mixed micelles display low cytotoxicity due to the degradability of the polymers. The in vivo images show that the high stability of the mixed micelles ensures a high tumor accumulation. This selective tumor accumulation results in an excellent inhibition of in vivo tumor growth and a high rate of apoptosis in cancerous tissues, with low toxicity. This highly stable, mixed micellar system with a pH-dependent drug release, which enables the precise delivery of drugs to the tumor lesions, is feasible to employ clinically in cancer therapy.

Low Bloom strength gelatin as a carrier for potential use in retinal sheet encapsulation and transplantation.

Retinal transplantation aims to restore vision for patients suffering from retinitis pigmentosa and age-related macular degeneration. Because the retinal sheets are fragile in nature, it is difficult to maintain graft integrity during surgical manipulation and after transplantation. In the present work, we report the feasibility of applying sandwich-like gelatin membranes as encapsulating carriers for retinal sheet transplantation applications. The relationship between the Bloom index of gelatin and the functionality of carrier membranes was studied by determinations of mechanical property, dissolution degree, melting point, cytocompatibility, biocompatibility, and transplant transfer and encapsulation efficiency. Irrespective of their Bloom strength, the gelatin membranes had a thickness sufficient to provide mechanical support for retinal sheets and would be beneficial to overcome the fragility of transplants during intraocular delivery. It was found that the lower the Bloom value of gelatin, the lower melting point of membranes. This allowed for easy fabrication of a stable sandwich-like encapsulating structure at 37 degrees C. The gelatins with lower Bloom strengths could possibly be dissolved to an extent required for the establishment of close contact between the retinal grafts and defective tissues. In addition, the carrier membranes made from the gelatins with low Bloom values showed a relatively higher cytocompatibility and biocompatibility as well as a higher transfer and encapsulation efficiency as compared to those with high Bloom values. It is concluded that the effect of Bloom index of gelatin plays a significant role in the membrane functionality and the gelatins with low Bloom values have substantial potential to be further developed as effective encapsulating carriers for the intraocular delivery of retinal sheets.

Blood-typing and irregular antibody screening through multi-channel microfluidic discs with surface antifouling modification.

A novel surface modification technology for microfluidic disks was developed for multichannel blood-typing detection and irregular antibody screening. The antifouling material, poly(ethylene glycol) methacrylate (PEGMA), was used to modify the surface of the microfluidic disk for improving its hydrophilicity and blood compatibility. With the modification of PEGMA, the hydrophilicity was sufficiently improved with a 44.5% reduction of water contact angle. The modified microfluidic disk also showed good biocompatibility with a reduction of hemolytic index (from 3.4% to 1.2%) and platelet adhesion (from 4.6 × 104/cm2 to 1.9 × 104/cm2). Furthermore, the PEGMA modification technique conducted on the microfluidic disk achieved successful adjustment of burst frequency for each chamber in the microchannel, allowing a sequential addiction of reagents in the test protocol of manual polybrene (MP) blood typing. Clinical studies showed that the proposed MP microfluidic disk method not only performed at extremely high consistency with the traditional tube method in the identification of ABO/RhD blood types, but also accomplished an effective screening method for detecting irregular antibodies. In conclusion, this study demonstrated that the easily mass-produced MP microfluidic disk exhibited good blood-typing sensitivity and was suitable for clinical applications.

Fabrication of redox-responsive Bi(mPEG-PLGA)-Se2 micelles for doxorubicin delivery.

Stimuli-responsive polymeric nanostructures have emerged as potential drug carriers for cancer therapy. Herein, we synthesized redox-responsive diselenide bond containing amphiphilic polymer, Bi(mPEG-PLGA)-Se2 from mPEG-PLGA and 3,3'-diselanediyldipropanoic acid (DSeDPA) using DCC/DMAP as coupling agents. Due to its amphiphilic nature, Bi(mPEG-PLGA)-Se2 self-assembled in to stable micelles in aqueous solution with a hydrodynamic size of 123.9 ±â€¯0.85 nm. The Bi(mPEG-PLGA)-Se2 micelles exhibited DOX-loading content (DLC) of 6.61 wt% and encapsulation efficiency (EE) of 54.9%. The DOX-loaded Bi(mPEG-PLGA)-Se2 micelles released 73.94% and 69.54% of their cargo within 72 h upon treatment with 6 mM GSH and 0.1% H2O2, respectively, at pH 7.4 and 37 °C. The MTT assay results demonstrated that Bi(mPEG-PLGA)-Se2 was devoid of any inherent toxicity and the DOX-loaded micelles showed pronounced antitumor activities against HeLa cells, 44.46% of cells were viable at maximum dose of 7.5 µg/mL. The cellular uptake experiment further confirmed the internalization of DOX-loaded Bi(mPEG-PLGA)-Se2 micelles and endowed redox stimuli triggered drug release in cytosol and nuclei of cancer cells. Overall, the results suggested that the smart, biocompatible Bi(mPEG-PLGA)-Se2 copolymer could serve as potential drug delivery biomaterial for the controlled release of hydrophobic drugs in cancer cells.

Tissue-engineered human corneal endothelial cell sheet transplantation in a rabbit model using functional biomaterials.

BACKGROUND: This study was performed to investigate whether transplantation of bioengineered human corneal endothelial cell (HCEC) sheet grafts into corneas denuded of endothelium could restore corneal function and clarity in a rabbit model. METHODS: After being labeled with PKH26 fluorescent dye, the adult HCECs derived from eye bank corneas were cultivated on the thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted surfaces for 3 weeks at 37 degrees C, and were harvested as transplantable cell sheets after incubation for 45 min at 20 degrees C. Attached by gelatin hydrogel discs, the bioengineered cell monolayers were transplanted to rabbit corneas denuded of endothelium (HCEC sheet group). Traumatized rabbit corneas were served as controls. Postsurgical corneas underwent clinical observations and histological examinations for 6 months. RESULTS: By transmission electron microscopy and Western blot analysis of zonula occludens-1 and Na+,K+ -adenosine triphosphatase proteins, the structure and function of HCEC sheets resembled those of native corneal endothelium. After endothelial cells were removed, corneas of each group turned severe edematous and opaque. In the HCEC sheet groups, corneal clarity was gradually restored and corneal thickness was significantly less than that in the control groups (P<0.05). The attached PKH26-positive HCECs spread on rabbit Descemet's membrane after receiving cell sheet grafts. Intraocular delivery of HCEC sheets by means of a minimally invasive technique (i.e., small-incision surgery using biodegradable hydrogels) demonstrated long-term graft integration with damaged corneas. CONCLUSIONS: These results indicate that using cultured HCECs and functional biomaterials, PNIPAAm and gelatin, an effective cell sheet-based therapy can be developed for the treatment of corneal endothelium deficiency.

Mixed micelles formed from graft and diblock copolymers for application in intracellular drug delivery.

A novel mixed micelle that comprised of poly(N-isopropylacrylamide-co-methacrylic acid)-graft-poly(D,L-lactide) (P(NIPAAm-co-MAAc)-g-PLA) with methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-b-PLA) was developed for application in cancer therapy. The mixed micelle had an multi-functional inner core of P(NIPAAm-co-MAAc)-g-PLA to enable intracellular drug delivery and an extended hydrophilic outer shell of mPEG to hide the inner core. Stability analysis of the mixed micelles in bovine serum albumin (BSA) solution indicates that the diblock copolymer mPEG efficiently protected the BSA adsorption on the mixed micelles because the hydrophobic groups of graft copolymer were efficiently screened by mPEG. From the drug release study, the mPEG-PLA diblock copolymer in mixed micelles slightly affected the functionalities of the P(NIPAAm-co-MAAc)-g-PLA graft copolymer; the graft copolymer still exhibited pH- and thermo-sensitivities in this core-shell structure. A change in pH deformed the structure of the inner core from that of aggregated P(NIPAAm-co-MAAc), causing the release of a significant quantity of doxorubicin (Dox) from mixed micelles. Clear differences between free Dox and Dox-mixed micelles were observed using confocal laser scanning microscopy (CLSM). This study presents not only a new micelle structure for a graft-diblock copolymer system, but also a method for overcoming some of the limitations on biomaterials used in intravenous injection.

Heterobifunctional poly(ethylene glycol)-tethered bone morphogenetic protein-2-stimulated bone marrow mesenchymal stromal cell differentiation and osteogenesis.

We describe a biomimetic mode of insoluble signaling stimulation to provide target delivery of bone morphogenetic protein-2 (BMP-2), with the aim of prolonging the retention of BMP-2 use in bone tissue engineering and to enable its localized release in response to cellular activity. In our novel localization process, we used heterobifunctional acrylate-N-hydroxysuccinimide poly(ethylene glycol) (PEG) as a spacer to tether BMP-2 onto a poly(lactide-co-glycolide) scaffold. Use of PEG-tethered BMP-2 was feasible because BMP-2 retained its activity after covalent conjugation. The PEG-tethered BMP-2 conjugate sustained stimulation and retained its mitogenic activity, notably affecting pluripotent stem cell proliferation and differentiation. We seeded the scaffolds with bone marrow-derived mesenchymal stromal cells as progenitor cells to evaluate their morphology and phenotypic expression. We also created bilateral, full-thickness cranial defects in rabbits to investigate the osteogenic effect of cultured mesenchymal stromal cells on bone regeneration in vivo. Histomorphometry and histology demonstrated that the PEG-tethered BMP-2 conjugate enhanced de novo bone formation after surgery. Our work revealed the potential for biomimetic surface engineering by entrapping signaling growth factor to stimulate osteogenesis. Our technique may provide a new platform for bone-engineered stem cell therapies.

Targeted delivery system for juxtacrine signaling growth factor based on rhBMP-2-mediated carrier-protein conjugation.

We propose a model of artificial juxtacrine signaling for the controlled release of recombinant human bone morphogenetic protein-2 (rhBMP-2) suitable for guided bone regeneration. A porous three-dimensional scaffold of poly-(lactide-co-glycolide) was fabricated by means of gel molding and particulate leaching. Collagen immobilization onto the scaffold surface was produced by performing photo-induced graft polymerization of acrylic acid, and rhBMP-2 was tethered to the collagenous surface by covalent conjugation. On pharmacokinetic analysis, in vitro enzyme-linked immunosorbent and alkaline phosphatase assays revealed sustained, slow release of rhBMP-2 over 28 days, with a cumulative release of one third of the initial load diffusing out of the scaffold. Conjugation of rhBMP-2 inhibited the free lateral diffusion and internalization of the activated complex of rhBMP-2 and the bone morphogenetic protein receptor. Osteoprogenitor cells were used as bone precursors to determine the expression of biosignaling growth factor in regulating cell proliferation and differentiation. To identify the phenotype of cells seeded on the rhBMP-2-conjugated scaffold, cellular activity was evaluated with scanning electron microscopy and with viability, histological, and immunohistochemical testing. The rhBMP-2-conjugated scaffold prolonged stimulation of intracellular signal proteins in cells. Enhancement of cell growth and differentiation was considered a consequence of juxtacrine signaling transduction. Animal studies of rhBMP-2-containing filling implants showed evidence of resorption and de novo bone formation. The present study revealed the potential of biomimetic constructs with co-immobilized adhesion and growth factors to induce osteoinduction and osteogenesis. Such constructs may be useful as synthetic bone-graft materials in orthopaedic tissue engineering.

A novel strategy for corneal endothelial reconstruction with a bioengineered cell sheet.

Cellular organization of foreign grafts constructed from cultivated cells is critical to successful graft-host integration and tissue repair. This study described a novel human corneal endothelial cell (HCEC) therapeutic method, where cultivated adult HCEC sheet with uniform orientation was prepared and transplanted to a rabbit cornea. Having a correct morphology and intact barriers, the HCEC sheet was made by the temperature-modulated detachment of monolayered HCECs from thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted surfaces and was delivered with proper polarity to the corneal posterior surface by a bioadhesive gelatin disc. Results of the in vivo studies, including the follow-up clinical observations and histological examinations, showed the laminated HCEC sheet was successfully integrated into rabbit cornea denuded with endothelial layer after the biodegradation of gelatin carrier. These data indicate the feasibility of the proposed procedure in cell therapy for corneal endothelial cell loss.

Bioengineered human corneal endothelium for transplantation.

OBJECTIVE: To investigate whether the bioengineered human corneal endothelial cell (HCEC) monolayers harvested from thermoresponsive culture supports could be used as biological tissue equivalents. METHODS: Untransformed adult HCECs derived from eye bank corneas were cultivated on a thermoresponsive poly-N-isopropylacrylamide-grafted surface for 3 weeks at 37 degrees C. Confluent cell cultures with a phenotype and cell density similar to HCECs in vivo were detached as a laminated sheet by lowering the culture temperature to 20 degrees C. In vitro characteristics of the HCEC sheets were determined evaluating their viability and by scanning electron microscopy, immunohistochemistry, and histological studies. RESULTS: After separation from culture surfaces via a thermal stimulus, the HCEC sheets remained viable. Polygonal cell morphology and multiple cellular interconnections were observed throughout the HCEC sheets. Immunolocalization of zonula occludens-1 and Na+,K+-adenosine triphosphatase (ATPase) indicated the formation of tight junctions and the distribution of ionic pumps at the cell boundary. In addition, we ascertained that cultured HCECs have a monolayered architecture that mimics native corneal endothelium. CONCLUSION: These data suggest that a well-organized and functional HCEC monolayer can feasibly be used as tissue equivalents for replacing compromised endothelium.Clinical Relevance Bioengineered human corneal endothelium fabricated from thermoresponsive supports can potentially offer a new therapeutic strategy for corneal endothelial cell loss.

Environmental-sensitive micelles based on poly(2-ethyl-2-oxazoline)-b-poly(L-lactide) diblock copolymer for application in drug delivery.

Anticancer drug doxorubicin (DOX) was physically loaded into the micelles prepared from poly(2-ethyl-2-oxazoline)-b-poly(L-lactide) diblock copolymers (PEOz-PLLA). PEOz-PLLA consists of hydrophilic segment PEOz and hydrophobic segment PLLA showed pH-sensitivity in the aqueous solution. The DOX-loaded micelle exhibited a narrow size distribution with a mean diameter around 170 nm. The micellar structure can preserve hydrophobic drug DOX under the physiological condition (pH 7.4) and selectively release DOX by sensing the intracellular pH change in late endosomes and secondary lysosomes (pH 4-5). At 37 degrees C, the cumulated released rate of DOX from micelles was about 65% at pH 5.0 in the initial 24 h. Additionally, polymeric micelles had low cytotoxicity in human normal fibroblast HFW cells for 72 h by using MTT assay. Moreover, DOX-loaded micelles could slowly and efficiency decrease cell viability of non-small-cell lung carcinoma CL3 cells. Taken together, PEOz-b-PLLA diblock polymeric micelles may act as useful drug carriers for cancer therapy.

Enzymatic degradation of PLLA-PEOz-PLLA triblock copolymers.

The enzymatic degradation of poly(L-lactide)-block- poly(2-ethyl-2-oxazoline)-block-poly(L-lactide) triblock copolymer (PLLA-PEOz-PLLA) was investigated using efficient enzyme proteinase K. PLLA-PEOz-PLLA solution-cast film lost a considerable amount of hydrophilic copolymers in the first 2 h, and the degradation after 2 h proceeded predominantly by surface erosion. The two faces of the hydrolyzed film exhibited different morphologies following enzymatic degradation. The lower face showed many spherulites, which are the superstructural morphology of polymer crystals. Porous spheres based on crystalline PLLA were observed on the upper face, because they were more resistant to enzymatic attack. The crystallinity of the films increased monotonously with the hydrolysis time, thus, the absorption of water gradually decreased. The analysis of degradation residues revealed that many colloids of poly(2-ethyl-2-oxazoline)-co-polyethylenimine (PEOz-co-PEI) copolymers were dispersed in the buffer solution. The average diameter, 1 microm, of the colloids was reduced to 200 nm by advanced degradation. The proteinase K exhibited remarkable hydrolysis not only at the ester bond but also the amide bond.

Preparation and characterization of intelligent core-shell nanoparticles based on poly(D,L-lactide)-g-poly(N-isopropyl acrylamide-co-methacrylic acid).

New thermo-responsive, pH-responsive, and biodegradable nanoparticles comprised of poly(D,L-lactide)-graft-poly(N-isopropyl acrylamide-co-methacrylic acid) (PLA-g-P(NIPAm-co-MAA)) were developed by grafting biodegradable poly(D,L-lactide) onto N-isopropyl acrylamide and methacrylic acid. A core-shell type nano-structure was formed with a hydrophilic outer shell and a hydrophobic inner core, which exhibited a phase transition temperature above 37 degrees C suitable for biomedical application. Upon heating above the phase transition temperature, PLA-g-P(NIPAm-co-MAA) nanoparticle showed a polarity increase of pyrene in either buffer solution or intra-hepato-carcinoma cells as determined by fluorescence measurement, indicating that the structure of nanoparticles caused leakages from outer shell copolymers aggregation and collapsed. The drug loading level of 5-fluorouracil (5-FU) encapsulated in the PLA-g-P(NIPAm-co-MAA) nanoparticles can be as high as 20%. The release of 5-FU from nanoparticles was strongly controlled by the pH in the aqueous solution. Based on these results, PLA-g-P(NIPAm-co-MAA) nanoparticles can be used as a drug carrier for intracellular delivery of anti-cancer drug.

Polymeric micelles with a pH-responsive structure as intracellular drug carriers.

Polymeric micelles based on poly(L-lactide)-b-poly(2-ethyl-2-oxazoline)-b-poly(L-lactide) (PLLA-PEOz-PLLA) ABA triblock copolymers were designed as intracellular drug carriers. The PLLA-PEOz-PLLA micelles adopt a "flower-like" arrangement with A-blocks at the core and a B-block on the shell under neutral condition. The deformation of the core-shell structure is then promoted by the aggregation of PEOzs due to the formation of inter- and intra-hydrogen bonding between protonated nitrogen and carbonyl groups. The experiments on in vitro release have confirmed that the release of doxorubicin (DOX) from micelles was successfully inhibited at pH 7.4. In contrast, an accelerated release of DOX from micelles was observed at acidic conditions. The results of growth inhibition assay indicated that the cell-killing rate of DOX-loaded micelles gradually approached that of free DOX as increasing the concentration and the incubation time. The overlay of fluorescent images on CLSM observation clearly demonstrated the colocalization of DOX with acidic compartments, suggesting that the drug release was successfully triggered in the acidic organelles by means of micelle deformation.

Preparation of controlled release ophthalmic drops, for glaucoma therapy using thermosensitive poly-N-isopropylacrylamide.

In this study, controlled release ophthalmic agents for glaucoma therapy were developed based on the thermosensitivity of poly-N-isopropylacrylamide (PNIPAAm). The clear solution of PNIPAAm was known to undergo phase transition when the temperature was raised from the room temperature to about 32 degrees C. The drug was entrapped in the tangled polymer chains or encapsulated within the crosslinked polymer hydrogel at room temperature, and released progressively after topical application (i.e., at a higher temperature). Linear PNIPAAm and crosslinked PNIAAm nanoparticles containing epinephrine were prepared. The drug release rate and cytotoxicity were investigated in vitro. Ophthalmic formulations based on either linear PNIPAAm or the mixture of linear PNIPAAm and crosslinked PNIPAAm nanoparticles were administered to rabbits and the intraocular pressure (IOP)-lowering effect was evaluated. The decreased pressure response of the formulation based on linear PNIPAAm lasted six-fold longer than that of the conventional eye drop. Furthermore, for formulation based on the mixture of linear PNIPAAm and crosslinked nanoparticles, the pressure-lowering effect lasted eight times longer. These results suggest the use of thermosensitive polymer solutions or hydrogels is potential in controlled release antiglaucoma ophthalmic drugs.