Biodegradation of amino-acid-based poly(ester amide)s: in vitro weight loss and preliminary in vivo studies

A systematic in vitro biodegradation study of regular poly(ester amide)s (PEAs) composed of naturally occurring hydrophobic alpha-amino acids, fatty diols and dicarboxylic acids using gravimetric (weight loss) method was carried out in the presence of hydrolases like alpha-chymotrypsin, lipase and a complex of proteases of Papaya. The last enzyme was used for modeling the catalytic action of nonspecific proteases. It was found that the PEAs, in the presence of enzyme solutions, were biodegraded by surface chemical erosion mechanism, according to the first-order kinetics. Spontaneous immobilization (adsorption) of the enzymes onto the PEAs surfaces was observed. The surface immobilized enzyme can also catalyze the chemical erosion of the PEAs. The enzymes could also be impregnated into the PEAs to make PEAs biochemically biodegraded, i.e. 'self-destructive', at a target rate. A comparison of in vitro biodegradation data of the PEAs with polylactide (PDLLA) showed that PEAs exhibited a far more tendency toward enzyme catalyzed biodegradation than PDLLA. A preliminary in vivo biodegradation study of selected PEA films in rats with and without lipase-impregnation showed that those PEAs were completely absorbed within 1-2 months post-implantation (for the lipase-impregnated ones) and 3-6 months (for the lipase-free ones). These findings prompt us to suggest that these new PEAs may have a great potential for designing drug sustained/controlled release devices, as well as implantable surgical devices.     

Drug release from starch-acetate microparticles and films with and without incorporated alpha-amylase

Acetylation of starch considerably decreases its swelling and enzymatic degradation. Thus, starch-acetate (SA) based delivery systems may be suitable for controlled drug delivery. The aim of the present study was to evaluate drug release from the SA microparticles (SA mps) and SA films. The average degree of acetyl substitution (DS) per glucose residue in the starch was either 1.9 (SA DS 1.9) or 2.6 (SA DS 2.6). Timolol (mw 332), calcein (mw 623) and bovine serum albumin (BSA, mw 68,000) were used as model drugs. A continuous timolol release from the both SA mps was observed in phosphate buffer solution (PBS) pH 7.4 (50-days incubation). The release of timolol was faster from the SA DS 1.9 mps than from the SA DS 2.6 mps. Calcein release from both SA mps was continuous in PBS pH 7.4 (5-days incubation). But, calcein release profile from the SA DS 2.6 film in PBS pH 7.4 showed discontinuities. However, the release of calcein from both SA films was continuous in human serum in vitro during the 7-day incubation, i.e. enzymes enhanced calcein release. Thus, alpha-amylase was incorporated into the SA films in order to enhance drug release from the films. However, the effects of incorporation of alpha-amylase on the model macromolecule (BSA) release from the SA films were modest. In conclusion, this study demonstrates the achievement of slow release of different molecular weight model drugs from the SA mps and films as compared to fast release from the native starch preparations. DS of SA, physicochemical properties of a drug and the presence of enzymes can all affect drug release profiles from SA based preparations.     

Novel soy protein wound dressings with controlled antibiotic release: mechanical and physical properties

Naturally derived materials are becoming widely used in the biomedical field. Soy protein has advantages over various types of natural proteins employed for biomedical applications due to its low price, non-animal origin and relatively long storage time and stability. In the current study soy protein isolate (SPI) was investigated as a matrix for wound dressing applications. The antibiotic drug gentamicin was incorporated into the matrix for local controlled release and, thus, protection against bacterial infection. Homogeneous yellowish films were cast from aqueous solutions. After cross-linking they combined high tensile strength and Young’s modulus with the desired ductility. The plasticizer type, cross-linking agent and method of cross-linking were found to strongly affect the tensile properties of the SPI films. Selected SPI films were tested for relevant physical properties and the gentamicin release profile. The cross-linking method affected the degree of water uptake and the weight loss profile. The water vapor transmission rate of the films was in the desired range for wound dressings (∼2300 g m⁻² day⁻¹) and was not affected by the cross-linking method. The gentamicin release profile exhibited a moderate burst effect followed by a decreasing release rate which was maintained for at least 4 weeks. Diffusion was the dominant release mechanism of gentamicin from cross-linked SPI films. Appropriate selection of the process parameters yielded SPI wound dressings with the desired mechanical and physical properties and drug release behavior to protect against bacterial infection. These unique structures are thus potentially useful as burn and ulcer dressings.     

Controlled release of ethacrynic acid from poly(lactide-co-glycolide) films for glaucoma treatment

Ethacrynic acid (ECA) is a potential glaucoma drug that can reduce intraocular pressure. However, conventional methods of ECA administration may cause toxicity to normal eye tissues and are inconvenient to patients. Therefore, we developed and characterized an ECA loaded poly(lactide-co-glycolide) (PLGA) copolymer film, and quantified the therapeutic efficacy of the film implanted in the rabbit eye. In the aqueous medium, the release of ECA from the PLGA50:50 film was time dependent and more than 90% of ECA was released within a week. This release profile was consistent with the kinetics of water uptake and microstructural changes of PLGA50:50 films as revealed by an electron microscopy examination. ECA release and PLGA degradation caused a gradual pH decrease in the release medium. The total pH decrease was 0.4 unit in 3 days. We also observed that the initial rate of ECA release was positively correlated with the weight ratio of ECA versus PLGA and inversely correlated with the molar ratio of lactide versus glycolide in PLGA films. At the end of a 3-day incubation, the cumulative release of ECA from PLGA50:50, PLGA85:15 and PLGA100:00 films were 78.8%, 9.35% and 3.60%, respectively. When the PLGA50:50 film loaded with ECA was implanted into the sclera of rabbit eyes, the intraocular pressure was significantly reduced and the reduction was maintained for at least 10 days. These data indicate that PLGA films have a potential to be used as a controlled ECA release device for glaucoma treatment.     

A vanadium/aspirin complex controlled release using a poly(beta-propiolactone) film. Effects on osteosarcoma cells.

A delivery system for vanadium was developed using poly(beta-propiolactone) (PbetaPL) films. The release kinetics of a complex of vanadium (IV) with aspirin (VOAspi) was evaluated with films prepared from polymers of different molecular weights, as well as with variable drug load. A sustained release of vanadium over 7 days was achieved. The drug release kinetics depends on contributions from two factors: (a) diffusion of the drug; and (b) erosion of the PbetaPL film. The experimental data at an early stage of release were fitted with a diffusion model, which allowed determination of the diffusion coefficient of the drug. VOAspi does not show strong interaction with the polymer, as demonstrated by the low apparent partition coefficient (approximately 10(-2)). UMR106 osteosarcoma cells were used as a model to evaluate the anticarcinogenic effects of the VOAspi released from the PbetaPPL film. VOAspi-PbetaPL film inhibited cell proliferation in a dose-response manner and induced formation of approximately half of the thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation. compared to that with free VOAspi in solution. The unloaded PbetaPL film did not generate cytotoxicity, as evaluated by cell growth and TBARS. Thus, the polymer-embedded VOAspi retained the antiproliferative effects showing lower cytotoxicity than the free drug. Results with VOAspi-PbetaPL films suggest that this delivery system may have promising biomedical and therapeutic applications.     

Poly(iminocarbonates) as potential biomaterials

Poly(iminocarbonates), based on diphenols such as hydroquinone or Bisphenol A were synthesized. Poly(iminocarbonates) degraded under physiological conditions. Compression moulded discs of poly-(hydroquinone-iminocarbonate) showed near zero-order release for low loadings (1%, w/w) of Eosin Y. Poly(Bisphenol A-iminocarbonate) formed transparent films and strong fibres. It is a tough polymer with high tensile strength. Under physiological conditions complete erosion of a thin film of poly(Bisphenol A-iminocarbonate) required about 200 d. The release profile for p-nitroaniline from solvent cast films of poly(Bisphenol A-iminocarbonate) was characterized by a lag period (20 d), followed by near zero-order release for 90 d. Initial toxicological studies revealed no inflammatory cell response after implantation in the corneas of rabbits or subcutaneously in mice.     

Novel gradient casting method provides high-throughput assessment of blended polyester poly(lactic-co-glycolic acid) thin films for parameter optimization

Pure polymer films cannot meet the diverse range of controlled release and material properties demanded for the fabrication of medical implants or other devices. Additives are added to modulate and optimize thin films for the desired qualities. To characterize the property trends that depend on additive concentration, an assay was designed which involved casting a single polyester poly(lactic-co-glycolic acid) (PLGA) film that blends a linear gradient of any PLGA-soluble additive desired. Four gradient PLGA films were produced by blending polyethylene glycol or the more hydrophobic polypropylene glycol. The films were made using a custom glass gradient maker in conjunction with a 180 cm film applicator. These films were characterized in terms of thickness, percent additive, total polymer (PLGA+additive), and controlled drug release using drug-like fluorescent molecules such as coumarin 6 (COU) or fluorescein diacetate (FDAc). Material properties of elongation and modulus were also accessed. Linear gradients of additives were readily generated, with phase separation being the limiting factor. Additive concentration had a Pearson's correlation factor (R) of >0.93 with respect to the per cent total release after 30 days for all gradients characterized. Release of COU had a near zero-order release over the same time period, suggesting that coumarin analogs may be suitable for use in PLGA/polyethylene glycol or PLGA/polypropylene glycol matrices, with each having unique material properties while allowing tuneable drug release. The gradient casting method described has considerable potential in offering higher throughput for optimizing film or coating material properties for medical implants or other devices.     

The study of improved controlled release of vincristine sulfate from collagen-chitosan complex film

A water-in-oil-in-oil double-emulsion solvent/evaporation method was used to prepare vincristine sulfate (VCR) loaded poly(lactide-co-glycolide) microspheres, and then VCR microspheres were mixed with collagen and (or) chitosan swelling solution and lyophilized to form polymeric films. The films were cross-linked by 0.3% glutaraldehyde (GA). Encapsulation efficiency and release kinetics of VCR microspheres were determined, as well as release kinetics and in vitro degradation of the film. The rate of VCR release from the film submerged in PBS (pH 6.8) and the content were measured by high-performance liquid chromatography (HPLC). The physichemical properties of the film, such as surface morphology, mechanical function, and differential scanning calorimetry, were also measured. VCR was released from the film in a prolonged period and the initial burst release of the film was less significant. In the degradation experiment, the film containing chitosan degraded more slowly than that without chitosan. The films comprising collagen and chitosan could achieve the release kinectics of a relatively constant release. It has a promising future.     

A vanadium/aspirin complex controlled release using a poly(β - propiolactone) film. Effects on osteosarcoma cells

A delivery system for vanadium was developed using poly(β-propiolactone) (PβPL)films. The release kinetics of a complex of vanadium (IV) with aspirin (VOAspi) was evaluated with films prepared from polymers of different molecular weights, as well as with variable drug load. A sustained release of vanadium over 7 days was achieved. The drug release kinetics depends on contributions from two factors: (a) diffusion of the drug; and (b) erosion of the PβPL film. The experimental data at an early stage of release were fitted with a diffusion model, which allowed determination of the diffusion coefficient of the drug. VOAspi does not show strong interaction with the polymer, as demonstrated by the low apparent partition coefficient (approximately 10-2). UMR106 osteosarcoma cells were used as a model to evaluate the anticarcinogenic effects of the VOAspi released from the PβPL film. VOAspi-PβPL film inhibited cell proliferation in a dose-response manner and induced formation of approximately half of the thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, compared to that with free VOAspi in solution. The unloaded PβPL film did not generate cytotoxicity, as evaluated by cell growth and TBARS. Thus, the polymer-embedded VOAspi retained the antiproliferative effects showing lower cytotoxicity than the free drug. Results with VOAspi-PβPL films suggest that this delivery system may have promising biomedical and therapeutic applications.     

Controlled degradation of multilayered poly(lactide-co-glycolide) films using electron beam irradiation

The ability to undergo predictable and controlled degradation allows biopolymers to release prescribed dosages of drugs locally over a sustained period. However, the bulk or homogeneous degradation of some of these polymers like poly(L-lactide) (PLLA) and poly(lactide-co-glycolide) (PLGA) work against a better controlled release of the drugs. Inducing the polymers to undergo surface erosion or layer-by-layer degradation could provide a better process of controlled drug release from the polymers. This study has demonstrated that surface erosion degradation of PLGA is possible with the use of a multilayer film system, with PPdlLGA [plasticized poly(D,L-lactide-co-glycolide) (PdlLGA)] as the surface layers and poly(L-lactide-co-glycolide) as the center layer. The use of the more hydrophilic PPdlLGA as the surface layer resulted in a faster degradation of the surface layers compared to the center layer, thus giving a surface erosion degradation effect. The rate of surface degradation could also be controlled with electron beam (e-beam) radiation, where e-beam irradiation was shown to alter the degradation time and onset of polymer mass loss. It was also shown that the more highly irradiated PPdlLGA surface layers had an earlier onset of mass loss, which resulted in a faster reduction in overall film thickness. The ability to control the rate of film thickness reduction with different radiation dose promises a better controlled release of drugs from this multilayer PLGA film system.     

Dual roles of hyaluronic acids in multilayer films capturing nanocarriers for drug-eluting coatings

We developed hyaluronic acid (HA)-based multilayer films capturing polymeric nanocarriers (NCs) for drug delivery. The electrostatic interactions between positively charged linear polyethylene imines (LPEI) and negatively charged HAs are the main driving forces to form multilayers based on the layer-by-layer (LbL) deposition. NCs were easily incorporated within the multilayer film due to intra- and/or inter-hydrogen bonding among HA chains. The amount of NCs captured by the HA chains was varied by the ratio between HAs and NCs as well as the length (i.e., molecular weight) and absolute number density of HAs in solution. Biocompatibility of the NC-capturing HA multilayer films was tested with the human dermal fibroblast (HDF) culture. In addition, the controlled release of paclitaxel (PTX) from the HA multilayer films successfully led to the apoptosis of human aortic smooth muscle cells (hSMC) in vitro, implying that the NC-capturing HA multilayer films would be quite useful as drug-eluting stent systems to prevent the restenosis after surgery.     

Soluble sustained release gene delivery system

Delivery of genetic substance to target cells remains an obstacle for efficient utilization of gene therapy approaches. In this study, we describe a formulation of methacrylate acid copolymer carrier of DNA, in which the release rate of the gene can be controlled by pH. Plasmid release was coupled with the polymer's dissolution, which was accelerated in alkali conditions. The released plasmid was intact and bioactive, although alteration from closed circular supercoil to relaxed conformation was observed. Confocal laser scanning microscopy detected the plasmid DNA along the central layers of the polymeric film. Gene delivery systems controlled by the dissolution of the polymeric films offer flexibility in quantity and size of the incorporated DNA, and therefore could have a potential for in vivo use.     

Bioerodible system for sequential release of multiple drugs

Because many complex physiological processes are controlled by multiple biomolecules, comprehensive treatment of certain disease conditions may be more effectively achieved by administration of more than one type of drug. Thus, the objective of the present research was to develop a multilayered, polymer-based system for sequential delivery of multiple drugs. The polymers used were cellulose acetate phthalate (CAP) complexed with Pluronic F-127 (P). After evaluating morphology of the resulting CAPP system, in vitro release of small molecule drugs and a model protein was studied from both single and multilayered devices. Drug release from single-layered CAPP films followed zero-order kinetics related to surface erosion of the association polymer. Release studies from multilayered CAPP devices showed the possibility of achieving intermittent release of one type of drug as well as sequential release of more than one type of drug. Mathematical modeling accurately predicted the release profiles for both single layer and multilayered devices. The present CAPP association polymer-based multilayer devices can be used for localized, sequential delivery of multiple drugs for the possible treatment of complex disease conditions, and perhaps for tissue engineering applications, that require delivery of more than one type of biomolecule.     

胶原/纤维蛋白胶复合药膜的性质及其体外抑瘤效应初步研究

目的 制备载硫酸长春新碱（VCR）微球的胶原/纤维蛋白胶的缓释药膜并研究其性质,考察京尼平的交联浓度（0、0.05、0.1、0.2 mol/ml）对药膜性质的影响.方法 冷冻干燥和京尼平交联法制备多孔胶原膜,将VCR微球混合纤维蛋白胶后加入多孔胶原膜中制备胶原/纤维蛋白胶复合药膜,考察药膜的表面形态、机械性能、降解性质和体外释药行为,并用CCK-8法检测药膜对3种肿瘤细胞（人肺癌细胞株A549、人乳腺癌细胞株MCF-7和人宫颈癌细胞株HeLa）的体外抑瘤率.结果 随着交联剂京尼平浓度的增加,药膜机械强度增加,而断裂伸长率降低.VCR微球与纤维蛋白胶及胶原复合制备成药膜,可达到双重缓释的作用,减少药物突释,并延缓药物释放.未交联的F0药膜药物24h突释为（41.8±3.4）％,京尼平交联后的F0.05、F0.1和F0.2药膜的药物突释分别为（38.4±4.1）％、（35.2±3.6）％和（34.3±3.7）％.未载药的F0.1药膜无显著细胞毒效应,而载药的F0.1药膜在不同时间释放的药液对3种肿瘤细胞株均有显著的抑制作用,且对各细胞株敏感度不同.结论 F0.1药膜能不同程度减少药物的突释,使药物释放更加平稳缓慢,有望用作抗肿瘤药物载体.     

Sustained-release delivery systems of triclosan for treatment of Streptococcus mutans biofilm

Dental diseases are chronic infections caused by oral bacteria harboring the dental biofilm. Local sustained-release delivery systems prolong the duration of a drug in the oral cavity, thus enhancing its therapeutic potential, while reducing its side effects. Triclosan is an agent that was found to have an antibacterial effect against oral bacteria. However, its substantivity in the oral cavity is low, resulting in reduced antibacterial efficiency. The purpose of this study was to develop a local sustained release device containing triclosan and to test its antibacterial efficacy on Streptococcus mutans biofilm. Our results show that we can formulate an ethylcellulose-based, nondegradable, sustained-release device in which 80% of the loaded triclosan is released over a 10-day period. The release rate of triclosan corresponded to the Higuchi's planar homogenous diffusion release model (r2 = 0.998). A degradable local sustained-release delivery based on a methacrylate ester matrix was also developed for a faster release rate of triclosan. The release kinetics in those types of sustained-release delivery systems was erosion control. The local sustained-release delivery system significantly affected the viability of S. mutans in biofilm compared to placebo as was tested by confocal laser scanning microscopy. Our in vitro results show that triclosan can be incorporated into degradable or nondegradable sustained-release drug delivery systems. The release of triclosan from the local sustained-release delivery system can be controlled, thus extending its antibacterial properties.     

The toxicological study of CCNU controlled release film in vivo

We planted the Lomustine (CCNU) controlled release films into normal mice, and after a period of time observed the effects of the films on the blood cells and the brain nerve cells of the mice. Compared with the traditional administration (PO), the results indicated that the planted CCNU controlled release film had less effects on the blood cells, and caused less harm to the brain nerve cells. So the conclusion was that the planted CCNU controlled release film had no significant acute arrest of bone marrow and neural toxicity.     

Electrochemical method for entrapment of oligonucleotides in polymer-coated electrodes.

Oligonucleotides were incorporated into conducting films of poly(3, 4-ethylene dioxythiophene) by an electrochemical method that involves two steps. The electrode is first coated with the polymer film by electropolymerization followed by electrooxidation of the formed polymer in the presence of the oligonucleotide. Neutral water soluble polymers such as poly(vinylpyrrolidone) or poly(ethylene glycol) were added to the polymerizing medium resulting in higher incorporation yields of oligonucleotides. The results showed that a payload of about 1.5 nmol/cm2 can be achieved in an 8 micron thick film for an oligomer concentration of about 70 microM in the working solution. Various physical methods were used to analyze the surface of the polymer-coated electrodes. Results showed the presence of domains of varying sizes at the film surface, corresponding to the insertion of the hydrophilic polymer within the matrix of the conductive polymer. The release of entrapped oligonucleotides from the coated film exhibited a three-step profile: a "burst" period during the first 5 min followed by an intermediate and a very slow release period lasting several days. Such polymer films could be exploited as useful reservoirs of biologically active substances for in vivo delivery to targeted tissues. For example, coated stents that can release antiproliferative agents such as antisense oligonucleotides at the site of balloon dilatation may be of interest in the treatment of postangioplasty restenosis.     

Controlled release of complexed DNA from polycaprolactone film: comparison of lipoplex and polyplex release

This report investigates the comparative in vitro controlled release and transfection efficiencies of pDNA-lipofectamine complex (lipoplex) and pDNA-poly(ethylene imine) complex (polyplex), from a biodegradable polycaprolactone (PCL) film. The effect of molecular weight of gelatin used as a porogen on in vitro release and transfection efficiency was also studied. A sustained release profile was obtained for naked pDNA and lipoplex from polymeric films for a month, while the release of polyplexes (PEI/DNA) is simply a burst at day 5, with little or no release thereafter. The release of polyplexes from PCL films is retarded due to interaction between the polyplexes and the polymer. A high burst release was seen for naked pDNA which was suppressed in the presence of gelatin. The extent of suppression of the burst effect by gelatin increased with its molecular weight. For complexed pDNA (lipoplex), the release was slow, but could be accelerated using gelatin; again the acceleration in release is dependant on the molecular weight of the gelatin used. The addition of gelatin as a porogen has no effect on the release of polyplexes from PCL films. The bioactivity of released plasmid DNA and complexes was studied by in vitro transfection using COS-7 cells. Transfection was observed from released lipoplexes samples till day 9 from PCL film with lower MW gelatin and till day 18 in the case of PCL films with higher MW gelatin. The results also showed that the bioactivity of released lipoplexes was superior to that of the naked pDNA.     

Perivascular graft heparin delivery using biodegradable polymer wraps

Heparin remains the gold-standard inhibitor of the processes involved in the vascular response to injury. Though this compound has profound and wide-reaching effects on vascular cells in culture and animal models, its clinical utility has been questionable at best. It is clear that the mode of heparin delivery is critical to its potential and it may well be that routine forms of administration are insufficient to observe benefit given the heparin's short half-life and complex pharmacokinetics. When ingested orally, heparin is degraded to inactive oligomer fragments while systemic administration is complicated by the need for continuous infusion and the potential for uncontrolled hemorrhage. Thus alternative heparin delivery systems have been proposed to maximize regional effects while limiting systemic toxicity. Yet, as heparin is such a potent antithrombotic compound and since existing local delivery systems lack the ability to precisely regulate release kinetics, even site-specific therapy is prone to bleeding. We now describe the design and development of a novel biodegradable system for the perivascular delivery of heparin to the blood vessel wall with well-defined release kinetics. This system consists of heparin-encapsulated poly(DL lactide-co-glycolide) (pLGA) microspheres sequestered in an alginate gel. Controlled release of heparin from this heterogeneous system could be obtained over a period of 25 days in vitro. The experimental variables affecting heparin release from these matrices were investigated. Gel permeation chromatography (GPC) and scanning electron microscopy (SEM) were used to monitor the degradation process and found to correlate well with the release kinetics. Heparin-releasing gels inhibited growth of bovine vascular smooth muscle cells in tissue culture in a dose-dependent manner. Moreover, gel release controlled vascular injury in denuding and interposition vascular graft animal models of disease even when uncontrolled bleeding was evident with standard matrix-type release. This system may therefore provide an effective means of examining the effects of various compounds in the control of smooth muscle cell proliferation in accelerated arteriopathies and also shed light on the biologic nature of these processes.