Water-soluble biodegradable cationic polyphosphazenes for gene delivery.

Polyphosphazenes bearing cationic moieties were synthesized from poly(dichloro)phosphazene, which in turn was obtained by thermal polymerization of hexachlorocyclotriphosphazene in 1,2,4-trichlorobenzene. Next, either 2-dimethylaminoethanol (DMAE) or 2-dimethylaminoethylamine (DMAEA) side groups were introduced by a substitution reaction. The polymers were purified by dialysis against water and tetrahydrofuran, lyophilized and evaluated as polymeric transfectants. The polyphosphazenes were able to bind plasmid DNA yielding positively charged particles (polyplexes) with a size around 80 nm at a polymer/DNA ratio of 3:1 (w/w). The polyphosphazene-based polyplexes were able to transfect COS-7 cells in vitro with an efficiency comparable to a well-known polymeric transfectant [poly(2-dimethylaminoethyl methacrylate), pDMAEMA]. The toxicity of both polyphosphazenes was lower than pDMAEMA. The transfection efficiency for the poly(DMAE)phosphazene-based polyplexes was about threefold higher in the absence of serum than in the presence of 5.0% fetal bovine serum. This is probably caused by unfavorable interactions of the polyplexes with serum proteins. In contrast, the poly(DMAEA)phosphazene-based polyplexes showed a threefold lower transfection activity in the absence of serum. For this system, serum proteins likely masked the toxicity of the polyplexes, as shown by the XTT cell viability assay and confocal laser scanning microscopy studies. Preliminary degradation studies indicate that the polymers were indeed degradable. The half-life at pH 7.5 and 37 degrees C was around 7 days for poly(DMAE)phosphazenes and 24 days for poly(DMAEA)phosphazenes. This study shows that polyphosphazenes are a suitable and promising new class of biodegradable polymeric carriers for gene delivery.     

New biodegradable polymers for injectable drug delivery systems.

Many biodegradable polymers were used for drug delivery and some are successful for human application. There remains fabrication problems, such as difficult processability and limited organic solvent and irreproducible drug release kinetics. New star-shaped block copolymers, of which the typical molecular architecture is presented, results from their distinct solution properties, thermal properties and morphology. Their unique physical properties are due to the three-dimensional, hyperbranched molecular architecture and influence microsphere fabrication, drug release and degradation profiles. We recently synthesized thermosensitive biodegradable hydrogel consisting of polyethylene oxide and poly(L-lactic acid). Aqueous solution of these copolymers with proper combination of molecular weights exhibit temperature-dependent reversible sol-gel transition. Desired molecular arrangements provide unique behavior that sol (at low temperature) form gel (at body temperature). The use of these two biodegradable polymers have great advantages for sustained injectable drug delivery systems. The formulation is simple, which is totally free of organic solvent. In sol or aqueous solution state of this polymer solubilized hydrophobic drugs prior to form gel matrix.     

Thermosensitive and biodegradable polymeric micelles for paclitaxel delivery.

The preparation, release and in vitro cytotoxicity of a novel polymeric micellar formulation of paclitaxel (PTX) were investigated. The micelles consisted of an AB block copolymer of poly(N-(2-hydroxypropyl) methacrylamide lactate) and poly(ethylene glycol) (pHPMAmDL-b-PEG). Taking advantage of the thermosensitivity of pHPMAmDL-b-PEG, the loading was done by simply mixing of a small volume of a concentrated PTX solution in ethanol and an aqueous polymer solution and subsequent heating of the resulting solution above the critical micelle temperature of the polymer. PTX could be almost quantitatively loaded in the micelles up to 2 mg/mL. By dynamic light scattering and cryo-transmission electron microscopy, it was shown that PTX-loaded micelles have a mean size around 60 nm with narrow size distribution. At pH 8.8 and 37 degrees C, PTX-loaded micelles destabilized within 10 h due to the hydrolysis of the lactic acid side group of the pHPMAmDL. Because the hydrolysis of the lactic acid side groups is first order in hydroxyl ion concentration, the micelles were stable for about 200 h at physiological conditions. The presence of serum proteins did not have an adverse effect on the stability of the micelles during at least 15 h. Interestingly, the dissolution kinetics of pHPMAmDL-b-PEG micelles was retarded by incorporation of PTX, indicating a strong interaction between PTX and the pHPMAmDL block. The PTX-loaded micelles showed a release of the incorporated 70% of PTX during 20 h at 37 degrees C and at pH 7.4. PTX-loaded pHPMAmDL-b-PEG micelles showed comparable in vitro cytotoxicity against B16F10 cells compared to the Taxol standard formulation containing Cremophor EL, while pHPMAmDL-b-PEG micelles without PTX were far less toxic than the Cremophor EL vehicle. Confocal laser-scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) analysis of fluorescently labelled micelles showed that pHPMAmDL-b-PEG micelles were internalized by the B16F10 cells. The present results suggest that pHPMAmDL-b-PEG block copolymer micelles are a promising delivery system for the parenteral administration of PTX.     

Branched biodegradable polyesters for parenteral drug delivery systems.

Continuous, 'infusion-like' drug release profiles from biodegradable parenteral delivery systems are difficult to achieve for proteins and other hydrophilic macromolecular drugs with commonly used linear polyesters from lactic acid (PLA) and its random copolymers with glycolic acid (PLG). Drug release rates can be modified either by increasing the hydrophilicity of polyesters or by manipulating the polymer architecture to adjust polymer degradation rates and thus drug release. Therefore, we investigated different branching concepts for biodegradable polyesters of PLA and PLG. For one four- and eight-arm poly(ethylene oxide)s (PEO) were grafted with shorter polyester chains leading to star-branched structures. Secondly we obtained comb-like polyesters using both charged and uncharged dextrans or poly(vinyl alcohol)s (PVA) as hydrophilic backbones. The star-shaped and brush-like grafted polymers were intensively characterized by methods, such as NMR, IR, SEC-SLS, DSC and viscosity measurements. Tailor-made properties make these novel biodegradable polyesters promising candidates for parenteral protein delivery systems. While the star-branched polyesters have shown some interesting properties with respect to their degradation behavior, retaining the PEO blocks longer than ABA triblock copolymers, their release properties need further optimization. Brush-like branched polyesters on the other hand seem to possess both degradation and release properties meriting further investigations for parenteral protein delivery systems.     

Enhanced cytosolic drug delivery using fully biodegradable poly(amino oxalate) particles

Rapid endosomal escape of drug carriers is crucial to enhancing the efficacy of their macromolecular payload, especially proteins that are susceptible to lysosomal degradation. In this paper, we report poly(amino oxalate) (PAOX) as a new protein delivery system that is capable of disrupting endosomes and mediating cytosolic drug delivery. A cationic fully-biodegradable PAOX was synthesized from a one-step reaction of oxalyl chloride, cyclohexanedimethanol and piperazinediethanol. The incorporation of tertiary amine groups in the backbone of PAOX enhanced its hydrolytic nature, which results in a fast drug release. The studies of confocal fluorescence imaging using calcein and LysoTracker Red revealed that PAOX particles disrupted endosomes via “proton sponge” effects and mediated the cytosolic delivery of membrane-impermeable calcein. A protein delivery efficiency of PAOX particles was evaluated using catalase as a model protein. Catalase-loaded PAOX microparticles significantly inhibited hydrogen peroxide generation in Phorbol-12-myristate-13-acetate (PMA)-stimulated macrophages, in a dose-dependent manner. Given the excellent biocompatibility and physicochemical properties, we anticipate that PAOX is a promising cytosolic protein delivery system and is useful for the treatment of acute inflammatory diseases.     

Sustained interferon-gamma delivery from a photocrosslinked biodegradable elastomer.

The application of protein therapeutics for long-term, localized delivery has been hindered by a lack of a delivery device that releases active protein at a concentration within their therapeutic window. A protein delivery system that uses an osmotic pressure delivery mechanism and a photocrosslinked biodegradable elastomer has been designed in an attempt to overcome this limitation. The elastomer is prepared through the UV initiated crosslinking of end terminal acrylated star-poly(epsilon-caprolactone-co-D,L-lactide). Interferon-gamma (IFN-gamma) was released from the optimum formulation at a constant rate of 23 ng/day over 21 days. A cell-based assay showed that over 83% of released IFN-gamma was bioactive. Furthermore, it was demonstrated that bovine serum albumin co-lyophilized with IFN-gamma was released at the same rate as IFN-gamma. This delivery formulation may be clinically useful for sustained, local protein drug delivery applications.     

Rational design of biodegradable cationic polycarbonates for gene delivery

Polycarbonates provide an attractive option for use as gene delivery vectors owing to their biocompatibility and ease of incorporating functional moieties. In this study, we described an approach to synthesize cationic polymers with well-defined molecular weights and narrow polydispersities by an organocatalytic ring-opening polymerization of functional cyclic carbonates containing alkyl halide side chains, followed by a subsequent functionalization step with bis-tertiary amines designed to facilitate gene binding and endosomal escape. The cationic polycarbonate effectively condensed DNA at low N/P ratios, generating nanoparticles (83 to 124 nm in diameter) with positive zeta potentials (~ 27 mV). In addition, reporter gene expression efficiencies in HepG2, HEK293, MCF-7 and 4T1 cell lines were high even in the presence of serum. Importantly, the polycarbonate delivery agent demonstrated minimal cytotoxicity at the optimal N/P ratios determined to confer high gene expression efficiencies. Therefore, this biodegradable polymer is presented as a promising non-viral vector for gene delivery.     

Acid-cleavable polymeric core-shell particles for delivery of hydrophobic drugs.

Here we describe the combined use of acid-labile microgel approach and RAFT-mediated seeded dispersion polymerization technique to prepare an acid-cleavable core-shell like polymeric colloidal system for the delivery of hydrophobic drugs at slightly acidic sites. A new bisacrylate acetal crosslinker was copolymerized with n-butyl acrylate (BA) in the presence of a RAFT agent using a dispersion polymerization technique, which yielded crosslinked spherical particles with the size ranging between 150 and 500 nm. The particles were cleaved in a pH-dependent manner similar to the acid-labile hydrolysis behaviour of the crosslinker. In order to mask the hydrophobic surface of the particles, polyethylene glycol acrylate (PEG-A) was grafted onto poly(BA) seed particles via the RAFT agent groups on the particle surface. The acidic-site selective delivery potential of the poly(BA)-g-poly(PEG-A) particles was assessed in-vitro using a lipophilic fluorescent dye as a model hydrophobic drug. Ca. 73% and 34% of the total dye loaded in the particles was found to be released at pH 5.0 and 7.4 in 24 h, respectively. The growth of human neuroblastoma cells was not affected by the incubation with the core-shell particles and their cleavage by-products up to 3 mg/ml concentration. The physicochemical and the functional features support the potential value of the acid-cleavable poly(BA) core-poly(PEG-A) shell particles as carriers for the delivery of hydrophobic drugs at acidic sites.     

Fabrication of biodegradable spheroidal microparticles for drug delivery applications

Particle shape, in addition to size, is becoming increasingly recognized as important in the design of drug carriers for in vivo use. However, few methods exist for fabricating non-spherical particles from biodegradable polymers. This work describes for the first time the fabrication of biodegradable spheroidal microparticles using the simple oil-in-water emulsion solvent evaporation technique (O/W ESE). Unloaded and paclitaxel-loaded spheroids were fabricated from poly(lactic-co-glycolic acid) (PLGA), and the shape and size of fabricated spheroids were manipulated by controlling fabrication process parameters including stir speed, aqueous and oil phase viscosity, aqueous phase pH, and the polymer molecular weight and end group. The presented data show that high aqueous phase viscosity, basic aqueous phase pH and hydrophilic polymer side chains and end groups are all conditions that favor the formation of spheroidal particles. The described technique is advantageous over methods currently described in the literature in its simplicity in setup, high particle yield and adaptability to a wide range of biodegradable polymers and therapeutics.     

Long-circulating DNA-complexed biodegradable multiblock copolymers for gene delivery: degradation profiles and evidence of dysopsonization.

Biodegradable cationic polymers have become promising alternatives to traditional polycationic gene delivery systems in which the high charge densities of high molecular weight polymers contribute significantly to cellular toxicities. Previous research has shown that biodegradable, multiblock copolymers (MBC), PEG-PLL-g-16% His, are efficient gene carriers with negligible cellular toxicities. The present research was designed to characterize the polymer degradation as well as to determine the biodistribution of the MBC after systemic administration. Polymer degradation was performed in buffer as a function of pH, in serum and within polymer/pDNA complexes. The MBC exhibited exponential decay with a half-life (t1/2) of approximately 14 min at pH 9.0, approximately 5 h at pH 7.4 and approximately 2 h in serum. However, there was little or no degradation observed at pH 4.0 and the MBC within the complexes degraded between 4 and 8 h in serum. Biodistribution data performed with fluorescently labeled polymer and pDNA revealed that intact complexes remained in the blood up to 3 days, which was also reflected in the organs as a function of time. Therefore, the cumulative data suggest that PEG may be sterically stabilizing complexes in vivo via dysopsonization in which serum proteins mask the complexes from elements of the reticuloendothelial system (RES).     

Disassembly of polyethylenimine-DNA particles in vitro: implications for polyethylenimine-mediated DNA delivery.

Here a simple in vitro assay was used to investigate the disassembly of nanoparticles of polyethylenimine (PEI) and DNA. Particles were formed with various PEIs, allowed to mature for 10 min, and then exposed to different competitors (RNA, DNA, BSA or heparin) or to different conditions of pH or osmolarity. DNA release was determined by gel electrophoresis or spectroscopy. The presence of heparin or high salt yielded complete particle disassembly for all PEIs tested. The addition of RNA to particles formed with linear PEIs or branched 2 kDa PEI resulted in rapid DNA release, but RNA induced only partial disassembly of particles formed with large branched PEIs. In the presence of competitor DNA, slow disassembly was observed with particles made with linear PEIs or branched 2 kDa PEI but not for particles made with larger branched PEIs. The presence of BSA resulted in partial disassembly of PEI-DNA particles, but acidic pH did not affect particle stability. If particles were allowed to mature longer than 10 min in NaCl, subsequent heparin-mediated DNA release decreased as the incubation time and the PEI:DNA ratio increased. However, particles that matured in culture medium were disassembled by heparin independently of maturation time or PEI:DNA ratio. It was concluded that branched PEIs have a higher affinity for DNA than linear PEIs, that the intracellular disassembly of PEI-DNA particles may involve interactions between PEI and cellular RNA, and that extended maturation of PEI-DNA particles in NaCl prior to transfection may limit the intracellular release of plasmid DNA.     

Microspheres for protein delivery prepared from amphiphilic multiblock copolymers. 1. Influence of preparation techniques on particle characteristics and protein delivery.

The entrapment of lysozyme in amphiphilic multiblock copolymer microspheres by emulsification and subsequent solvent removal processes was studied. The copolymers are composed of hydrophilic poly(ethylene glycol) (PEG) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks. Direct solvent extraction from a water-in-oil (w/o) emulsion in ethanol or methanol did not result in the formation of microspheres, due to massive polymer precipitation caused by rapid solvent extraction in these non-solvents. In a second process, microspheres were first prepared by a water-in-oil-in-water (w/o/w) emulsion system with 4% poly(vinyl alcohol) (PVA) as stabilizer in the external phase, followed by extraction of the remaining solvent. As non-solvents ethanol, methanol and mixtures of methanol and water were employed. However, the use of alcohols in the extraction medium resulted in microspheres which gave an incomplete lysozyme release at a non-constant rate. Complete lysozyme release was obtained from microspheres prepared by an emulsification-solvent evaporation method in PBS containing poly(vinyl pyrrolidone) (PVP) or PVA as stabilizer. PVA was most effective in stabilizing the w/o/w emulsion. Perfectly spherical microspheres were produced, with high protein entrapment efficiencies. These microspheres released lysozyme at an almost constant rate for approximately 28 days. The reproducibility of the w/o/w emulsion process was demonstrated by comparing particle characteristics and release profiles of three batches, prepared under similar conditions.     

Design of novel bioconjugates for targeted drug delivery.

This paper summarizes recent work on the design and development of targeted polymeric bioconjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. Polymerizable antibody Fab' fragment (MA-Fab') has been developed and used in the preparation of targeted HPMA copolymer-mesochlorin e6 conjugates for the treatment of human ovarian carcinomas. The reactivity of the MA-Fab' in copolymerization with HPMA depended on the length of the spacer between the monomer double bond and the antibody Fab' fragment. The biological activity of the antibody Fab' fragment was maintained after incorporation into the HPMA copolymer. Novel aromatic azo spacers were designed and incorporated into HPMA copolymer-drug (cyclosporin A, 9-aminocamptothecin) conjugates for the colon-specific drug delivery and for the treatment of colon diseases. The colon-specific drug release from the conjugates was controlled by the structures of both drug and spacers. Lectins, wheat germ agglutinin (WGA) and peanut agglutinin (PNA), were conjugated to the colon-specific polymer drug conjugates to enhance specific adhesion onto colon tissues.     

心肌内双层多孔生物可降解性药物缓释支架的制备

目的：制备心肌内双层多孔生物可降解性药物缓释支架,评估其对透室壁性心肌血管重建术后心肌孔道的作用效果。方法：以聚己内酯为材料,以牛血清白蛋白为模型药物,以聚乳酸-聚乙醇酸共聚物为药物载体,制备成生物可降解性药物缓释支架。采用考马斯亮蓝试剂法对支架上牛血清白蛋白含量及体外释放量进行测定,万能材料测定仪测定支架的力学性能。制备猪慢性心肌局部缺血模型,体内评估该支架在透室壁性心肌血管重建术后对心肌孔道的作用效果。结果：该支架牛血清白蛋白携带量为每支架10mg,30d后牛血清白蛋白释放量达80%,支架压缩80%时承受的应力为1.2MPa,在透室壁性心肌血管重建后可保持心肌孔道通畅。结论：成功制备心肌内双层多孔生物可降解性药物缓释支架,能承受心肌压力并达到缓慢控制释放药物的效果,可维持透室壁性心肌血管重建后的心肌孔道通畅。     

Biodegradable microspheres for protein delivery

In a very short time, since their emergence, the field of controlled delivery of proteins has grown immensely. Because of their relatively large size, they have low transdermal bioavailabilities. Oral bioavailability is generally poor since they are poorly absorbed and easily degraded by proteolytic enzymes in the gastrointestinal tract. Ocular and nasal delivery is also unfavorable due to degradation by enzymes present in eye tissues and nasal mucosa. Thus parenteral delivery is currently most demanding and suitable for delivery of such molecules. In systemic delivery of proteins, biodegradable microspheres as parenteral depot formulation occupy an important place because of several aspects like protection of sensitive proteins from degradation, prolonged or modified release, pulsatile release patterns. The main objective in developing controlled release protein injectables is avoidance of regular invasive doses which in turn provide patient compliance, comfort as well as control over blood levels. This review presents the outstanding contributions in field of biodegradable microspheres as protein delivery systems, their methods of preparation, drug release, stability, interaction with immune system and regulatory considerations.