The Antimicrobial Effects of Colistin Encapsulated in Chelating Complex Micelles for the Treatment of Multi-Drug-Resistant Gram-Negative Bacteria: A Pharmacokinetic Study.

Background: Infections caused by multi-drug-resistant Gram-negative bacteria (MDR-GNB) are an emerging problem globally. Colistin is the last-sort antibiotic for MDR-GNB, but its toxicity limits its clinical use. We aimed to test the efficacy of colistin-loaded micelles (CCM-CL) against drug-resistant Pseudomonas aeruginosa and compare their safety with that of free colistin in vitro and in vivo. Materials and methods: We incorporated colistin into chelating complex micelles (CCMs), thus producing colistin-loaded micelles (CCM-CL), and conducted both safety and efficacy surveys to elucidate their potential uses. Results: In a murine model, the safe dose of CCM-CL was 62.5%, which is much better than that achieved after the intravenous bolus injection of 'free' colistin. With a slow drug infusion, the safe dose of CCM-CL reached 16 mg/kg, which is double the free colistin, 8 mg/kg. The area under the curve (AUC) levels for CCM-CL were 4.09- and 4.95-fold higher than those for free colistin in terms of AUC0-t and AUC0-inf, respectively. The elimination half-lives of CCM-CL and free colistin groups were 12.46 and 102.23 min, respectively. In the neutropenic mice model with carbapenem-resistant Pseudomonas aeruginosa pneumonia, the 14-day survival rate of the mice treated with CCM-CL was 80%, which was significantly higher than the 30% in the free colistin group (p < 0.05). Conclusions: Our results showed that CCM-CL, an encapsulated form of colistin, is safe and effective, and thus may become a drug of choice against MDR-GNB.

Improved photodynamic cancer treatment by folate-conjugated polymeric micelles in a KB xenografted animal model.

Photodynamic therapy (PDT) is a light-induced chemical reaction that produces localized tissue damage for the treatment of cancers and various nonmalignant conditions. In the clinic, patients treated with PDT should be kept away from direct sunlight or strong indoor lighting to avoid skin phototoxicity. In a previous study, it was demonstrated that the skin phototoxicity of meta-tetra(hydroxyphenyl)chlorin (m-THPC), a photosensitizer used in the clinic, can be significantly reduced after micellar encapsulation; however, no improvement in antitumor efficacy was observed. In this work, a folate-conjugated polymeric m-THPC delivery system is developed for improving tumor targeting of the photosensitizer, preventing photodamage to the healthy tissue, and increasing the effectiveness of the photosensitizers. The results demonstrate that folate-conjugated m-THPC-loaded micelles with particle sizes around 100 nm are taken up and accumulated by folate receptor-overexpressed KB cells in vitro and in vivo, and their PDT has no significant adverse effects on the body weight of mice. After an extended delivery time, a single dose of folate-conjugated m-THPC-loaded micelles has higher antitumor effects (tumor growth inhibition = 92%) through inhibition of cell proliferation and reduction of vessel density than free m-THPC or m-THPC-loaded micelles at an equivalent m-THPC concentration of 0.3 mg kg(-1) after irradiation. Furthermore, folate-conjugated m-THPC-loaded micelles at only 0.2 mg kg(-1) m-THPC have a similar antitumor efficacy to m-THPC or m-THPC-loaded micelles with the m-THPC concentration at 0.3 mg kg(-1) , which indicates that the folate conjugation on the micellar photosensitizer apparently reduces the requirement of m-THPC for PDT. Thus, folate-conjugated m-THPC-loaded micelles with improved selectivity via folate-folate receptor interactions have the potential to reduce, not only the skin photosensitivity, but also the drug dose requirement for clinical PDT.

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.

Development of 68Ga-glycopeptide as an imaging probe for tumor angiogenesis.

Objective. This study was aimed to study tissue distribution and tumor imaging potential of (68)Ga-glycopeptide (GP) in tumor-bearing rodents by PET. Methods. GP was synthesized by conjugating glutamate peptide and chitosan. GP was labeled with (68)Ga chloride for in vitro and in vivo studies. Computer outlined region of interest (counts per pixel) of the tumor and muscle (at the symmetric site) was used to determine tumor-to-muscle count density ratios. To ascertain the feasibility of (68)Ga-GP in tumor imaging in large animals, PET/CT imaging of (68)Ga-GP and (18)F-FDG were conducted in New Zealand white rabbits bearing VX2 tumors. Standard uptake value of tumors were determined by PET up to 45 min. To determine blood clearance and half-life of (68)Ga-GP, blood samples were collected from 10 seconds to 20 min. Results. Radiochemical purity of (68)Ga-GP determined by instant thin-layer chromatography was >95%. Tumor uptake values (SUV) for (68)Ga-GP and (18)F-FDG in New Zealand white rabbits bearing VX2 tumors were 3.25 versus 7.04. PET images in tumor-bearing rats and rabbits confirmed that (68)Ga-GP could assess tumor uptake. From blood clearance curve, the half-life of (68)Ga-GP was 1.84 hr. Conclusion Our data indicate that it is feasible to use (68)Ga-GP to assess tumor angiogenesis.

Reduced skin photosensitivity with meta-tetra(hydroxyphenyl)chlorin-loaded micelles based on a poly(2-ethyl-2-oxazoline)-b-poly(d,l-lactide) diblock copolymer in vivo.

Photodynamic therapy (PDT) is a light-induced chemical reaction that produces localized tissue damage for the treatment of cancers and other nonmalignant conditions. The activation of photosensitizers in a target tissue is accomplished with a specific light source in the presence of molecular oxygen. In the clinic, patients treated with PDT should be kept away from direct sunlight or strong indoor lighting to avoid skin phototoxicity. In this study, a photosensitizer encapsulated within a micelle was developed to overcome this problem. The pH-sensitive micelles were successfully incorporated with meta-tetra(hydroxyphenyl)chlorin (m-THPC), and the cytotoxicity and antitumor effects were investigated in vitro and in vivo. Our results demonstrated that PDT with m-THPC-loaded micelles had no significant adverse effects on the body weight of mice in vivo. Furthermore, after an extended delivery time, m-THPC-loaded micelles and free m-THPC had similar antitumor effects, but the m-THPC-loaded micelles had less skin phototoxicity. Thus, this strategy could be used as a potential nanocarrier for PDT-mediated cancer therapy.

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.

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.

Development of in situ thermosensitive drug vehicles for glaucoma therapy.

The goal of this research was to design thermosensitive drug vehicles for glaucoma therapy. Thermosensitive ophthalmic drop was prepared by mixing linear poly(N-isopropylacrylamide-g-2-hydroxyethyl methacrylate) (PNIPAAm-g-PHEMA), PNIPAAm-g-PHEMA gel particles and antiglaucoma drug. This produced polymeric eyedrop containing the drug epinephrine was a clear solution at room temperature which became a soft film after contacting the surface of cornea. The drug entrapped within the tangled polymer chains was therefore released progressively after topical application. Evaluation of the drug release responded as a function of crosslinking density and PHEMA macromer contents. The in vivo studies indicated that the intraocular pressure (IOP)-lowering effect for a polymeric eyedrop lasted for 26 h, which is significantly better than the effect of traditional eyedrop (8 h). Hence our investigations successfully prove that the thermosensitive polymeric eyedrop with ability of controlled drug release exhibits a greater potential for glaucoma therapy.

Development of polyion complex micelles for encapsulating and delivering amphotericin B.

A block copolymer poly(2-ethyl-2-oxazoline)-block-poly(aspartic acid) (PEOz-b-PAsp) was synthesized and investigated as the carrier of antifungal drug amphotericin B (AmB). Polyion complex (PIC) micelles with clear core-shell structures were identified by TEM, which revealed that the PAsp segment became hydrophobic after it interacted with AmB. PEOz-b-PAsp increased not only the solubility of AmB but also simultaneously the drug potency. The prolonged release of AmB from micelles effectively inhibited the growth of Candida albicans even after three days of administration. Moreover, the in vitro cytotoxicity of AmB-loaded micelles was less than that of Fungizone, which is a powerful antifungal antibiotic that is adopted to treat various fungal infections. The PEOz-b-PAsp PIC micelles with lower cytotoxicity and higher potency than Fungizone represent a potential means of encapsulating basic/amphoteric drugs.

Nonviral gene carriers based on diblock copolymers of poly(2-ethyl-2-oxazoline) and linear polyethylenimine.

Diblock copolymers that consist of poly(2-ethyl-2-oxazoline) (PEOz) and linear polyethylenimine (LPEI) were prepared for use as nonviral gene carriers. The PEOz-b-LPEI copolymers were synthesized by coupling PEOz with LPEI in a thiol-disulfide exchange reaction between the sulfhydryl and pyridyl disulfide terminal groups. A polymer/DNA weight ratio (P/D) of over 12 was required to enable PEOz-b-LPEI to condense DNA completely. The DNA-condensing capability of the diblock copolymers was increased with increasing the hydrolytic degrees of the LPEI segment. The PEOz-b-LPEI polyplexes were stable in 150 mM NaCl aqueous solution and had a mean diameter around 190 nm, whereas BPEI and LPEI polyplexes formed large aggregates in the range 300-500 nm. In addition, these polyplexes exhibited the sensitivity to solution pH and were dissociated in the acidic buffers (pH < or = 5.5). The results of in vitro cell viability and luciferase assay indicated that PEOz-b-LPEI showed not only low cytotoxicity but also high transfection efficiency in gene expression.

Polymer-DNA hybrid nanoparticles based on folate-polyethylenimine-block-poly(L-lactide).

The ability of amphiphilic block copolymers that consist of polyethylenimine (PEI) and poly(L-lactide) (PLLA) to modulate the delivery of plasmid DNA was evaluated. Folate-polyethylenimine-block-poly(l-lactide) (folate-PEI-PLLA) was synthesized by linking folic acid and PLLA to PEI diamine. Water-soluble polycation PEI provides gene-loading capability. Additionally, PEI is considered to exhibit high transfection efficiency and endosomal disrupting capacity. Hydrophobic PLLA that is incorporated into the gene delivery vector is believed to enhance the cell interactions and tissue permeability of the delivery system. Polymeric carrier containing folic acid is expected to be able to identify tumor surface receptors and transfect cells by receptor-mediated endocytosis. The results of agarose retardation assay indicated that the folate-PEI-PLLA began to form polyplexes at a polymer/DNA weight ratio (P/D) of over 10, whereas branched polyethylenimine (B-PEI) formed polyplexes with DNA at a ratio of above 1. The spherical particle morphology was supplemented with a particle size of approximately 100 nm at 10 P/D ratio. The results indicated that folate-PEI-PLLA with proper PEI/PLLA ratio effectively reduced cytotoxicity and maintained acceptable transfection efficiency. Low cytotoxicity of the folate-PEI-PLLA gives an advantage to high-dose administration.

New amphiphilic poly(2-ethyl-2-oxazoline)/ poly(L-lactide) triblock copolymers.

A new series of cationic, thermo-sensitive, and biodegradable poly(L-lactide)-poly(2-ethyl-2-oxazoline)-poly(L-lactide) (PLLA-PEOz-PLLA) triblock copolymers were synthesized by ring-opening polymerization. With increasing molecular weight and crystallinity of hydrophobic PLLA blocks, the critical micellization concentrations (CMC) occurred at lower concentration. The PLLA-PEOz-PLLA aqueous solution was transparent at room temperature. Heating the solution resulted in precipitations, which were caused by the combination of dehydration of water around PEOz and the aggregations of PLLA segments. Acid/base titration profiles indicated that PLLA-PEOz-PLLA were protonated at neutral and acidic conditions. Considerable buffering capacity was found over the entire pH range. The specific PLLA-PEOz-PLLA triblock copolymers with thermal- and pH-sensitive properties can be tailored by varying the compositions and can be applied as controlled release carries for biomedical applications.